Other cytokines involved in the evolution of brain infarction and contributing to aggravate neurological functions are IL-17 and IL-23 (Shichita et al., 2009; Konoeda et al., 2010; Ma et al., 2013; Swardfager et al., 2013). IL-17A secreted by γδ T cells promotes neutrophil recruitment and its blockade with specific antibodies exerts neuroprotection (Gelderblom et al., 2012). Despite the presence of IL-17A-positive lymphocytes in autoptic brain of stroke patients, IL-17 blood level does not seem to be a good predictor of stroke outcome as compared to other cytokines such as IL-6 (Zeng et al., 2013). In fact, plasma and cerebrospinal fluid levels of IL-6 correlate with stroke severity and poor clinical outcome in patients (Smith et al., 2004; Waje-Andreassen et al., 2005; Whiteley et al., 2009) and reduced blood concentrations of this cytokine have been correlated to the improved outcome induced by treatment with IL-1ra (Emsley et al., 2005). By contrast, in stroke animal models, IL-6 appears to play a neuroprotective role (Matsuda et al., 1996; Loddick et al., 1998; Herrmann et al., 2003), suggesting that its rapid and persistent elevation in the ischemic brain (Suzuki et al., 1999; Ali et al., 2000) may represent a compensatory mechanism to counteract the damaging effects of the insult. In fact, by activating its receptor and the downstream phosphorylation of STAT-3, IL-6 may induce neuroprotection (Yamashita et al., 2005; Jung et al., 2011). Moreover, IL-6 enhances the effectiveness of cell transplantation therapy in ischemic stroke, by reprogramming neural stem cells to tolerate oxidative stress and to induce angiogenesis through STAT-3 activation (Perini et al., 2001).

### Anti-Inflammatory Cytokines

While plasma concentrations of detrimental cytokines are elevated, levels of IL-10, associated with better outcome, are decreased in patients (Perini et al., 2001; Vila et al., 2003; Basic Kes et al., 2008). Results from animal models demonstrate that IL-10 represents a major downregulator of the detrimental effects of proinflammatory mediators during stroke and modulates neuronal vulnerability to excitotoxic ischemic damage (Spera et al., 1998; Grilli et al., 2000; Frenkel et al., 2005).

Another cytokine playing beneficial effects in cerebral ischemia is TGF-β (Gliem et al., 2012). Levels of this cytokine are elevated in the blood of patients 1 day after ischemic stroke (Yan et al., 2012) and in activated astrocytes and microglia/macrophages of the ischemic brain for at least 1 week in animal models (Lehrmann et al., 1998; Yamashita et al., 1999; Doyle et al., 2010). In rats, administration of a TGFβ antagonist aggravates brain damage caused by focal cerebral ischemia (Ruocco et al., 1999); whereas, in mice, intranasal delivery of this cytokine after stroke reduces infarct volume and increases neurogenesis in the subventricular zone (Ma et al., 2008). The beneficial effects of TGF-β involve both antiinflammatory effects, including inhibition of brain elevation of MCP-1 and MIP-1α (Pang et al., 2001), but also induction of glial scar formation (Logan et al., 1994) and anti-apoptotic effects (Zhu et al., 2002).

In mice, preconditioning with low dose LPS is associated with upregulation of anti-inflammatory cytokines, including TGF-β in brain and IL-10 in blood (Vartanian et al., 2011).

### Chemokines

In patients with acute ischemic stroke, serum levels of stromal cell-derived factor (SDF)-1α have been correlated with favorable long-term outcome (Kim et al., 2012) and this chemokine has been suggested to be a predictor of future stroke (Schutt et al., 2012). SDF-1α promotes bone marrow-derived cell targeting to the ischemic brain and improves local cerebral blood flow (Cui et al., 2007; Shyu et al., 2008), thus representing a promising target to implement stem cell therapy in patients. In fact, in the injured hemisphere, it activates CXCR4 on neural progenitor cells guiding their specific migration to the site of damage (Robin et al., 2006). Moreover, SDF-1α participates to the regulation of post-ischemic inflammation and it is involved in neurovascular repair (Wang et al., 2012). Despite these documented beneficial effects, some detrimental roles have also been suggested to be mediated by this chemokine, since pharmacological blockade of CXCR4 is neuroprotective in stroke by reducing BBB damage and inflammatory processes (Huang et al., 2013). Soluble fractalkine (CX3CL1) released by neurons upon an ischemic insult controls leukocyte trafficking and participates to the activation and chemoattraction of microglia to the injured brain via the activation of CX3CR1 receptors (Tarozzo et al., 2002; Dénes et al., 2008; Zhu et al., 2009). The detrimental effects of this chemokine are mediated by IL-1β and TNF-α in stroke mouse models (Soriano et al., 2002; Dénes et al., 2008). By contrast, in ischemic stroke patients, higher plasma concentrations of fractalkine are associated with better outcome and with low levels of systemic markers of inflammation (Donohue et al., 2012). In fact, by inhibiting caspase-3 and by activating adenosine receptors, exogenous administration of fractalkine is neuroprotective in wild-type rodents undergone permanent ischemia (Cipriani et al., 2011; Rosito et al., 2014). The apparent discrepancies between this latter finding and those produced in transgenic animals may be explained by the altered microglia responsiveness to fractalkine in the absence of constitutive fractalkine-CX3CR1 signaling (Cipriani et al., 2011).

Among the chemokines implicated in cerebral ischemia, MCP-1 (also known as CCL2) and MIP-1α have been shown to play an important role in promoting tissue damage via recruitment of inflammatory cells (Wang et al., 1995; Che et al., 2001; Takami et al., 2001; Minami and Satoh, 2003). The mRNA levels of both chemokines are elevated in the ischemic brain of rodents and MCP-1 levels are also increased in the cerebrospinal fluid of stroke patients (Losy and Zaremba, 2001). Mice lacking MCP-1 or its receptor, CCR2, display reduced infarct volume along with impaired leukocyte recruitment and reduced expression of inflammatory mediators in the injured brain (Hughes et al., 2002; Dimitrijevic et al., 2007; Strecker et al., 2011; Schuette-Nuetgen et al., 2012). Conversely, overexpression of MCP-1 prompts exacerbation of brain injury and increased cerebral recruitment of inflammatory cells (Chen et al., 2003). Moreover, MCP-1, as well as SDF-1α, promotes migration of newly formed neuroblasts from neurogenic regions to ischemic damaged areas (Robin et al., 2006; Yan et al., 2007).

Up-regulation of MCP-1 has been shown to underlie hypoxic preconditioning-induced stroke tolerance in mice (Stowe et al., 2012; Wacker et al., 2012); whereas, activation of CCR2 is involved in the neuroprotective effects exerted by both ischemic preconditioning and post-conditioning in mice subjected to global cerebral ischemia (Rehni and Singh, 2012).

Monocyte chemotactic protein-induced protein 1 (MCPIP1) deficiency exacerbates ischemic brain damage by upregulation of proinflammatory cytokines and this Zn finger-containing immunoregulatory protein also participates in LPS- and electroacupuncture-induced ischemic stroke tolerance (Liang et al., 2001; Jin et al., 2013).

### Enzymes: MMPs, COX, and NOS

MMPs play a crucial role in the evolution of the inflammatory response to ischemic brain injury (Cunningham et al., 2005). Stroke-induced damage to the BBB and hemorrhagic transformation are both induced by the activation of the gelatinases, MMP-2 and MMP-9, as demonstrated in animal models (Romanic et al., 1998; Rosenberg et al., 1998; Asahi et al., 2000) and in patients (Horstmann et al., 2003; Rosell et al., 2006, 2008).

Cerebral expression and activity of gelatinases increase very early after an ischemic insult, with a specific cellular expression pattern dependent on the spatio-temporal evolution of the damage (Rosenberg et al., 1998; Planas et al., 2001; Yang et al., 2007; Amantea et al., 2008). Pharmacological inhibition of gelatinases, as well as gene deletion of MMP-9, reduces infarct volume caused by focal cerebral ischemia in rodents (Romanic et al., 1998; Asahi et al., 2000; Gasche et al., 2001; Amantea et al., 2007, 2014b). The mechanisms by which gelatinases contribute to ischemic brain damage include disruption of BBB integrity, hemorrhagic transformation and white matter myelin degradation (Cunningham et al., 2005).  Moreover, the degradation of tight junction proteins claudin-5 and occludin by MMPs prompts hemorrhagic transformation, suggesting that these enzymes may represent a promising target for reducing the hemorrhagic complications associated with thrombolytic therapy (Yang and Rosenberg, 2011; Liu et al., 2012).

The up-regulation of the extracellular MMP inducer (EMMPRIN) occurring in peri-ischemic regions 2–7 days after focal ischemia in mice is coincident with the delayed increase of MMP-9, suggesting its involvement in neurovascular remodeling (Zhu et al., 2008). In fact, the late activation of MMP-9 promotes vascular endothelial growth factor (VEGF) signaling, contributing to neuronal survival and new vessels formation (Zhao et al., 2006). Thus, MMPs play a dual role in stroke injury, including early detrimental effects and beneficial roles at later stages after the insult. Moreover, MMPs participate to the protective response evoked by several preconditioning stimuli.  Similarly, the neuroprotective action of TNF-α induced by pre-ischemic physical exercise has been demonstrated to occur via reduced MMP-9 activity and amelioration of BBB dysfunction in rats subjected to transient MCAo, through the involvement of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation (Guo et al., 2008b; Chaudhry et al., 2010). Reduced activity of gelatinases was also associated with the neuroprotective effects exerted by hyperbaric oxygen preconditioning in hyperglycemic rats subjected to MCAo (Soejima et al., 2013).

After an ischemic insult, the expression of COX-2 is elevated in neurons, vascular cells and neutrophils, as demonstrated both in stroke patients and in animal models (Nogawa et al., 1997; Iadecola et al., 1999, 2001; Chakraborti et al., 2010). This enzyme contributes to post-ischemic inflammation through the production of toxic prostanoids and superoxide, and its deficiency or pharmacological inhibition leads to reduce BBB damage and to lower cerebral infiltration of leukocytes in rodent models of ischemic stroke (Iadecola et al., 2001; Candelario-Jalil et al., 2007). COX-2-derived prostaglandin E2 may also contribute to ischemic cell damage by disrupting Ca2+ homeostasis in neurons through activation of EP1 receptors (Kawano et al., 2006). Interestingly, an association between functional outcome and specific COX-2 variants has recently been demonstrated in ischemic stroke patients (Maguire et al., 2011).

#### TABLE 2 | Acute ischemic stroke trials for the clinical validation of anti-inflammatory and immunomodulatory drugs.

COX-2 has been implicated in hyperbaric oxygen preconditioning, since pharmacological inhibition of this enzyme abolishes the beneficial effects of the conditioning stimulus in a rat model of transient global cerebral ischemia (Cheng et al., 2011). Similarly, COX-2 induction participates to ischemic tolerance induced by cortical spreading depression (Horiguchi et al., 2006) or by a brief ischemic episode in rats (Choi et al., 2006; Pradillo et al., 2009), likely via the stimulation of the PGE2/PI3K/Akt pathway (Park et al., 2008). The elevated expression of COX-2 induced by ischemic preconditioning has been suggested to occur via a cascade involving epsilon protein kinase C and ERK1/2 activation, as well as NFkB nuclear translocation, as demonstrated in vitro (Kim et al., 2007, 2010).

While elevated expression of inducible NOS is associated with ischemic (Cho et al., 2005), TLR4-mediated (Pradillo et al., 2009) and anesthetic preconditioning (Kapinya et al., 2002), induction of this enzyme has also been implicated in the release of toxic amounts of NO by infiltrating neutrophils, microglia/macrophages and endothelial cells (Nakashima et al., 1995; Iadecola et al., 1996; Forster et al., 1999; Garcia-Bonilla et al., 2014b). NO released by endothelial cells early after the ischemic insult plays a beneficial role by inducing vasodilatation; whereas, at later stages, overactivation of neuronal NOS and, more importantly, de novo expression of inducible NOS contribute to ischemic tissue damage (Iadecola et al., 1996, 1997; Moro et al., 2004; Murphy and Gibson, 2007). In fact, excessive production of NO by inducible NOS is cytotoxic by promoting NFκB activation, by inhibiting ATP-producing enzymes, by producing peroxynitrite and by stimulating other pro-inflammatory enzymes such as COX-2 (Nogawa et al., 1998; Greco et al., 2011). Moreover, NO contributes to ischemic cell death via S-nitrosylation and, thereby, activation of glutamate receptor (GluR)-6 signaling (Yu et al., 2008) and MMP-9 (Gu et al., 2002).

NO plays a crucial role in cortical spreading depressioninduced tolerance to transient focal cerebral ischemia in rats (Horiguchi et al., 2005). Moreover, recent findings have demonstrated the involvement of endothelial NOS in remote ischemic preconditioning (Peng et al., 2012), while neuronal NOS has been involved in the neuroprotection exerted by remote post-conditioning (Pignataro et al., 2013).

# Concluding Remarks

Although ischemic stroke is a major cause of mortality and long-term disability worldwide (Go et al., 2014), current therapeutic approaches for its acute treatment only rely on blood flow restoration by thrombus lysis or removal (Mangiafico and Consoli, 2014; Berkhemer et al., 2015; Hacke, 2015). The therapeutic window, intended as the temporal range during which the endovascular treatment may reach the target of a useful recanalization, is conventionally set at a threshold of 6 h; therefore, only less that 10% of patients may actually benefit from these procedures. Thus, the identification of novel targets that allow widening the time-window for pharmacological intervention, as well as the possibility of limiting the development of ischemic brain damage by promoting innate beneficial responses, is a urgent challenge. To date, the clinical translation of immunomodulatory drugs has been hampered by the fact that most strategies tested in humans were purely based on antiinflammatory approaches (**Table 2**), disregarding the beneficial

# References

roles of some elements of the immune reaction to stroke injury (Amantea et al., 2014a). In this context, targeting immune responses that evolve during hours or days after the ischemic insult, by selectively promoting their beneficial components, represents a promising avenue for the development of more effective and safe stroke therapeutics.

# Author Contributions

All the authors participated in the collection, review, and analysis of the relevant literature, as well as to drafting and revising of the manuscript.

# Acknowledgments

This work was partially funded by the Italian Ministry of University and Research (PRIN prot. 20089 BARSR\_004 to GB), the University of Calabria (ex quota 60%) and the Italian Ministry of Health (Ricerca Corrente 2013 to GM and CT). This work was also partially supported by grants from Spanish Ministry of Economy And Competitiveness SAF2012- 33216 (MM), CSD2010-00045 (MM) and SAF2014-52225 (IL), from Fondo Europeo de Desarrollo Regional (FEDER) RETICS RD12/0014/0003 (IL), and from Regional Madrid Government S2010/BMD-2336 (MM) and S2010/BMD-2349 (IL).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Amantea, Micieli, Tassorelli, Cuartero, Ballesteros, Certo, Moro, Lizasoain and Bagetta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# **NF-**κ**B in innate neuroprotection and age-related neurodegenerative diseases**

*Annamaria Lanzillotta1 , Vanessa Porrini 1,2 , Arianna Bellucci 1 , Marina Benarese1 , Caterina Branca1 , Edoardo Parrella1 , Pier Franco Spano1,2 and Marina Pizzi 1,2 \**

*1 Department of Molecular and Translational Medicine, National Institute of Neuroscience, University of Brescia, Brescia, Italy, 2 IRCCS, San Camillo Hospital, Venice, Italy*

NF-κB factors are cardinal transcriptional regulators of inflammation and apoptosis, involved in the brain programing of systemic aging and in brain damage. The composition of NF-κB active dimers and epigenetic mechanisms modulating histone acetylation, finely condition neuronal resilience to brain insults. In stroke models, the activation of NF-κB/c-Rel promotes neuroprotective effects by transcription of specific anti-apoptotic genes. Conversely, aberrant activation of NF-κB/RelA showing reduced level of total acetylation, but site-specific acetylation on lysine 310, triggers the expression of proapoptotic genes. Constitutive knockout of c-Rel shatters the resilience of substantia nigra (SN) dopaminergic (DA) neurons to aging and induces a parkinsonian like pathology in mice. c-rel*−*/*−* mice show increased level of aberrantly acetylated RelA in the basal ganglia, neuroinflammation, accumulation of alpha-synuclein, and iron. Moreover, they develop motor deficits responsive to L-DOPA treatment and associated with loss of DA neurons in the SN. Here, we discuss the effect of unbalanced activation of RelA and c-Rel during aging and propose novel challenges for the development of therapeutic strategies in neurodegenerative diseases.

#### **Keywords: NF-**κ**B, epigenetic drugs, BDNF, c-Rel deficient mice, RelA (K310)**

# **Introduction**

In the central nervous system, NF-κB transcription factor acts as a pleiotropic regulator of target genes controlling physiological function (1) as well as pathological processes associated with neurodegeneration (2, 3).

The NF-κB family of transcription factors is composed by five different members that are p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). The RelA subunit, composing the activated p50/RelA dimer, and its post-transcriptional modifications play a pivotal role in the onset of neurodegenerative processes triggered by ischemic insults as well as glutamate or betaamyloid toxicity (4–8). The c-Rel subunit within activated NF-κB dimers counteracts the ischemic injury acting as an innate mechanism of neuroprotection (9). The c-Rel factor is reduced in neurons exposed to oxygen–glucose deprivation (OGD), and its over-expression can limit the cell death.

#### *Edited by:*

*Giuseppe Pignataro, Federico II University of Naples, Italy*

#### *Reviewed by:*

*Mariagrazia Grilli, University of Piemonte Orientale, Italy Markus Schwaninger, University of Lübeck, Germany*

#### *\*Correspondence:*

*Marina Pizzi, Department of Molecular and Translational Medicine, Section of Pharmacology, School of Medicine, University of Brescia, V. le Europa, 11, Brescia 25123, Italy<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology*

> *Received: 30 December 2014 Accepted: 21 April 2015 Published: 20 May 2015*

#### *Citation:*

*Lanzillotta A, Porrini V, Bellucci A, Benarese M, Branca C, Parrella E, Spano PF and Pizzi M (2015) NF-*κ*B in innate neuroprotection and age-related neurodegenerative diseases. Front. Neurol. 6:98. doi: 10.3389/fneur.2015.00098*

**Abbreviations:** 1B/DMT1, 1B isoform of divalent metal transporter-1; AMPK, AMP-activated kinase; BDNF, brain-derived neurotrophic factor; BIRC3, baculoviral IAP repeat-containing protein 3; DA, dopaminergic; HAT, histone acetyltransferase; HDAC, histone deacetylase; K310, lysine 310; MCAO, middle cerebral artery occlusion; MnSOD, superoxide dismutase; MPP+, 1-methyl-4-phenylpyridinium; NAD+, nicotinamide adenosine dinucleotide; OGD, oxygen–glucose deprivation; PD, Parkinson's disease; RIP1, receptor interacting protein; ROS, reactive oxygen species; SIRT1, sirtuin 1; SN, substantia nigra; SNc, SN pars compacta; TH, tyrosine hydroxylase; TNF, tumor necrosis factor; UCP4, mitochondrial uncoupling protein 4.

Moreover, the deficiency of c-Rel induces an age-related behavioral parkinsonism in mice, with degeneration of nigral dopaminergic (DA) neurons and development of a Parkinson's disease (PD)-like neuropathology (10). Recent evidence has shown that activation of NF-κB drives the systemic and brain aging process in mice (11, 12). Notably, Tilstra and colleagues demonstrated that RelA is the most contributing subunit in degenerative changes associated with senescence in a progeroid mice model (13).

We propose that while RelA activation accompanies normal brain aging, a misbalance between RelA and c-Rel might drive pathological aging by affecting the survival of substantia nigra (SN) DA cell and turning old mice into a parkinsonian phenotype.

# **RelA and c-Rel: Two Opposing Regulators of Neuronal Resilience to Brain Ischemia**

In the central nervous system, NF-κB factors are key players of a number of physiological processes such as neurogenesis (14), neuritogenesis (15), synaptic plasticity, learning, and memory (16–18). In recent years, a body of data has shown that NF-κB dysregulation participates to neurodegenerative mechanisms that occur in brain exposed to trauma or ischemia (19, 20), as well as in the brain of patients affected by PD (21, 22) and Alzheimer's disease (23).

The neuronal response to external stimuli relies on a differential activation of NF-κB dimers. We found that targeting RelA or c-Rel expression by antisense oligonucleotides (5) or siRNAs (6, 9) produces opposite effects on neuron survival. While over-activated p50/RelA dimers contribute to the apoptotic program, the c-Rel containing dimers increase the resilience of injured neuronal cells (**Figure 1**). Neurotoxic stimuli, such as ischemia (4, 9), glutamate (5), β-amyloid (7, 24), or 1-methyl-4-phenylpyridinium (MPP+) (25, 26), induce p50/RelA dimers activation and the transcription of a panel of pro-apoptotic genes (4). Conversely, c-Relcontaining dimers are responsible for anti-apoptotic gene expression by signals promoting neuroprotection in diverse neurotoxic settings, such as S100B in models of NMDA-mediated excitotoxicity (27), agonists at mGlu5 receptors against β-amyloid- (7) and MPP+ toxicity (25) or adipocyte-derived hormone leptin in neurons exposed to OGD (28). Over-expression of c-Rel in cultured neurons promotes anti-apoptotic effects by inducing the transcription of manganese superoxide dismutase (MnSOD) and Bcl-xL (7, 29, 30). c-Rel overabundance also limits the generation

**FIGURE 1 | The p50/RelA and p50/c-Rel dimers regulate neuronal survival**. Anti-apoptotic effects of NF-κB can be mediated by c-Rel containing dimers, which enhance neuronal resilience to oxidative stress by inducing Bcl-xL, MnSOD, UCP4, and BIRC3 expression. NF-κB anti-apoptotic effects can also be mediated by p50/RelA during preconditioning ischemia through the transcription of Bcl-xL. The pro-apoptotic effect elicited by NF-κB p50/RelA dimer in noxious ischemia is dependent on changes in the RelA acetylation state. A lower grade of RelA general acetylation, respect to control condition, but a site-specific acetylation on Lys 310 residue addresses the NF-κB binding toward Bim, Noxa, and DMT1 promoter.

of reactive oxygen species (ROS) by inducing transcription of the mitochondrial uncoupling proteins 4 (UCP4) (31), a brainspecific mitochondrial ion channel producing mild reduction of mitochondrial membrane potential and neuroprotection (32). Moreover, c-Rel can control the expression of baculoviral IAP repeat-containing protein 3 (BIRC3), named also as cIAP2, an anti-apoptotic E3 ligase, which can modulate the receptor interacting protein 1 (RIP1) activity by ubiquitination (33). Depending on its ubiquitination status, RIP1 can dictates if tumor necrosis factor (TNF)-α induces cell survival (and inflammation), or cell death pathways (34). Ubiquitinated RIP1 can recruit other kinases and finally induce NF-κB-mediated transcription of prosurvival and pro-inflammatory genes (TNF-α-dependent NF-κB activation) (35).

The elucidation of the dual effects of NF-κB activation on neuron survival was more evident in studies of severe brain ischemia. The activation of p50/RelA rapidly occurs in neurons and glial cells and has been implicated in pathogenesis of postischemic injury (36–38). In brain ischemic tissue of mice subjected to permanent middle cerebral artery occlusion (MCAO) and in primary cortical neurons exposed to OGD, NF-κB followed a similar pattern of activation (8, 39) characterized by increased nuclear translocation of p50/RelA dimer (4, 36) and decreased translocation of c-Rel-containing dimers (9). In these conditions, NF-κB activity was associated with an unbalance expression of pro-apoptotic RelA target genes, i.e., an increased expression of the pro-apoptotic members of Bcl-2 family genes (4) and reduced level of the anti-apoptotic member Bcl-xL (9, 40). During brain ischemia, RelA induced the expression of the 1B isoform of the divalent metal transporter-1 (1B/DMT1), the membrane carrier responsible for iron accumulation and brain damage after injury (41). The RelA-induced 1B/DMT1 expression acted as an upstream mechanism responsible for iron accumulation and contributing to neuronal cell death. While the over-expression of RelA increased cell death, the over-expression of c-Rel prevented neuronal loss in cortical neurons exposed to OGD, by increasing the transcription of Bcl-xL gene (9, 39). Knocking-down c-Rel expression exacerbated neuronal susceptibility to OGDmediated damage. Under brain ischemia, mice deficient for the c-Rel factor appeared insensitive to neuroprotective activity of leptin, a c-Rel inducer capable to limit cortical damage in wildtype mice (24, 28). These data strongly suggested that inhibition of c-Rel-containing dimers and activation of p50/RelA are key events in the pathogenesis of post-ischemic brain injury. In spite of these premises, p50/RelA activation *per se* appeared to be insufficient to drive pro-apoptotic transcription during brain ischemia. A similar pattern of p50/RelA nuclear translocation was found in mice exposed to a brief preconditioning ischemia (8) generating brain tolerance to a subsequent lethal ischemic injury (42). In neuronal cells, likewise in tumor cells, gene targeting by p50/RelA is finely regulated by post-transcriptional modification of RelA subunit, such as phosphorylation and acetylation (43). These modifications shape the strength and specificity of the NF-κB–DNA binding and final transcriptional responses. On this line, we investigated whether the activation of the p50/RelA dimer, in preconditioning or in lethal ischemia, differs in the RelA acetylation state (44).

# **RelA Acetylation is a Dynamic Process Which Tunes p50/RelA-Mediated Pro-Apoptotic Transcription in Brain Ischemia and is Modulated by Epigenetic Drugs**

Acetylation is the key post-translational modification of histones that controls the accessibility of chromatin to the transcriptional machinery and plays an essential role in gene activation (45). Lysine acetylation is reversible and controlled by the opposing activities of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

Besides histones, diverse non-histone proteins, including transcription factors NF-κB, are modified by HAT co-activators and HDACs (46). Acetylation of RelA on specific lysine residues (K122, 123, 218, 221, and 310) is a dynamic process that differently affects the RelA interaction with IκBα, the DNA-binding ability and the transcriptional activity of the protein (43, 47).

Members of class I HDACs, particularly HDAC1, HDAC2, and HDAC3, inhibited by vorinostat and entinostat (MS-275) are the most responsible for the general deacetylation of RelA (43, 48). Conversely, sirtuin 1 (SIRT1), an atypical class III HDAC that requires nicotinamide adenosine dinucleotide (NAD+) rather than zinc as a co-factor (49), activated by resveratrol, selectively deacetylates RelA at lysine 310 (K310) residue (8, 50).

Our studies have shown that mechanisms affecting the acetylation state of RelA might discriminate between protective and neurotoxic activation of NF-κB during ischemia (8). Protective ischemic preconditioning and harmful ischemia induced similar levels of p50/RelA activation, but only the ischemic injury induced an atypical RelA acetylation. The RelA that translocated to the nucleus in primary cortical neurons exposed to preconditioning OGD, or in cortices of mice subjected to preconditioning MCAO, showed a general deacetylation that paralleled the deacetylation on the K310 residue. Conversely, RelA activated in neurons exposed to lethal OGD or in cortices of mice subjected to noxious ischemia displayed a general deacetylation, but sitespecific acetylation on the K310 residue. This suggested that a mismatch between general and "site-specific" (K310) acetylation of RelA – where total acetylation decreases and K310 acetylation increases – could be responsible for pro-apoptotic transcription in ischemic conditions (8). The relevance of K310 acetylation to RelA-mediated effects during ischemia was demonstrated by mutagenesis analysis. The substitution of lysine with arginine at the RelA 310 residue impaired the acetylation at this site. In cells expressing the mutated RelA subunit, the OGD-mediated DMT1 transcription and the cell damage were totally prevented (8, 41). By undergoing such aberrant acetylation, RelA detached from the anti-apoptotic *Bcl-xL* promoter to bind the pro-apoptotic *Bim* promoter (44). In addition to changing the acetylation state of RelA, lethal ischemia produced a significant reduction of H3 histone acetylation (44), in line with previous evidence (51).

Prompted by these findings and in order to correct altered acetylation of RelA and histones after brain ischemia, we studied the association of the specific class I HDAC inhibitor MS-275 (52), and resveratrol (53). MS-275 is a synthetic benzamide derivative that currently is under clinical evaluation for cancer therapy (54). MS-275 has been shown to inhibit HDAC 1-3 with excellent pharmacokinetic properties (52).

Resveratrol is a polyphenol with multiple activities, including anti-oxidant, anti-tumorigenic, and neuroprotective activity (53, 55). In various models of brain ischemia, resveratrol delayed axonal degeneration after injury and mitigated the formation of free radical species as well as mitochondria-mediated apoptosis (56–59).

Widely known mechanisms of resveratrol action include the activation of the longevity factors SIRT1 (60) and AMP-activated kinase (AMPK), a serine–threonine kinase that acts as a key metabolic and stress sensor/effector (61). We found that treatments with either MS-275 or resveratrol in the post-ischemic period of mice subjected to MCAO decreased the infarct volume and displayed a significant neuroprotective activity in cortical neurons exposed to OGD (44). What's more, we showed that the combination of MS-275 and resveratrol at sub-threshold doses elicited a synergistic effect leading to maximal neuroprotection in both the animal and the cellular models of brain ischemia. MS-275 at the highest concentration tested, 1 μM, increased acetylation of H3 histones on K9/18 residues in neurons exposed to OGD. Resveratrol, unable to modify *per se* the H3 acetylation, produced a synergistic acetylation of H3 K9/18 when used in combination with MS-275.

Notably, the synergistic effect produced by co-administration of low doses of MS-275 (0, 1 μM) and resveratrol (3 μM) was sustained by AMPK activation by resveratrol. This could be ascribed to the fact that AMPK can activate many catabolic pathways to produce ATP and acetyl-CoA (62), the fundamental co-factor for HAT activity. AMPK has also been found to indirectly support the resveratrol-dependent SIRT1 induction by inducing NAD+ generation (61). As a consequence of AMPK-mediated enhancement of HAT and SIRT1 activity, the combination of MS-275 and resveratrol reversed the mismatch of RelA acetylation state in neurons exposed to OGD by, respectively, enhancing the RelA general acetylation and by reducing the acetylation at the K310 residue. The neuroprotective effect and transcription of antiapoptotic genes observed following the treatment with the drug combination appeared closely related to the restored optimal RelA acetylation state (8, 63). The protective and transcriptional effects produced by resveratrol and MS-275 in cortical neurons were entirely reproduced in the mouse MCAO model. The combination of sub-threshold doses of the drugs, administered during the reperfusion period, elicited a synergistic effect that limited the cerebral infarct volume and the subsequent neurological deficits. MS-275 and resveratrol in combination showed a long-lasting efficacy as the beneficial effects were still evident 72 h after the injury. Moreover, they displayed a wide therapeutic window as their efficacy was evident when administered within 7 h after the ischemic onset. The treatment induced a transcriptional switch from pro- to anti-apoptotic genes. The RelA binding shifted from the *Bim* to the *Bcl-xL* promoter and the acetylation of associated histones changed accordingly. H3 acetylation decreased at the *Bim* and increased at the *Bcl-xL* gene.

Recently, we evaluated the acetylation of histone residues at the brain-derived neurotrophic factor (BDNF) IV promoter in primary mouse cortical neurons exposed to OGD and treated with the synergistic combination of MS-275 and resveratrol (64).

We focused on promoter IV, which is known to be important for synaptic plasticity, both during neuronal development and in the adult brain (65). In the cortex, the promoter IV-dependent BDNF transcription accounts for the majority of the neuronal activity-induced BDNF expression (66, 67). Several studies have proposed BDNF as possible mediators of the beneficial effects of HDAC inhibitors in nervous system disorders (68–70). A ChIP analysis in cortical neurons showed that histones at the BDNF promoter IV were deacetylated after OGD exposure. Treatment in the post-OGD period with the combination of MS-275 and resveratrol significantly increased acetylation at H3K9/18 and H4K12 histones (64). These histone modifications may act cooperatively and possibly in parallel to other histone modifications to increase BDNF expression.

It can be proposed that neuroprotection elicited by MS-275 and resveratrol treatment is also closely related to modulation of BDNF expression and may improve neurologic function by enhancing neuronal plasticity.

All together, these data provide evidence that a pharmacological intervention targeting the epigenetic machinery can represents a promising strategy to limit post-ischemic injury with an extended therapeutic window.

# **c-Rel Deficiency Causes a Progressive Late-Onset Parkinsonism in Mice**

Following the evidence that RelA and c-Rel play opposing effects on neuron survival, and prevalence of p50/RelA activation versus p50/c-Rel triggers apoptotic cell death in brain ischemia (9), we tested whether a constitutive defect in c-Rel protein might affect the brain aging. Behavioral and pathological analyses of c-Rel knockout mice were performed at 2, 12, and 18 months of age and c-rel*−*/*−* mice showed to develop a PD-like syndrome and pathology with aging (10). Besides c-Rel subunit, other NF-κB subunits have been previously investigated for possible correlation with the onset of a parkinsonian pathology.  This increase was evident both in neuronal and glial cells of the SN, suggesting a role of RelA activation in neuronal cell loss and neuroinflammatory response associated with PD progression. The role of the other NF-κB subunits in PD remains unclear. The p50*−*/*−* mice treated with MPTP did not behave differently from wild-type mice (72), suggesting no, or only minor, role for p50 in the regulation of SN neuron resilience.

In 18-month-old c-rel*−*/*−* mice, the analysis of tyrosine hydroxylase (TH)-positive cells of the SN pars compacta (SNc) revealed a loss of DA neurons that paralleled the total loss of Nissl-stained cells. No significant change in the estimated number of DA cells was evident in 2- or 12-month-old c-rel*−*/*−* mice compared to age-matched controls. Notably, the loss of SNc DA neurons was associated with a decrease of TH-positive fibers and reduction of dopamine transporter (DAT) and dopamine content in the striatum. The 18-month-old c-rel*−*/*−* mice displayed no significant degeneration in the other brain areas examined, the nucleus basalis magnocellularis and the medial septal area, or in the ventral tegmental area that is generally spared in PD. Additional examination of SNc in aged c-rel*−*/*−* mice revealed a marked immunoreactivity for α-synuclein, the main protein constituent of Lewy bodies and Lewy neuritis and the key pathological feature of PD (73). Of note, fibrillary aggregated α-synuclein, detected by Thioflavin-S labeling, was present in the spared DA neurons of the SNc. Accumulation of insoluble α-synuclein in the mesencephalon was confirmed by the presence of a monomeric α-synuclein in the urea/SDS extracts used to solubilize the insoluble fraction. Neuroinflammation characterized by chronic microglial reactivity, RelA activation, and iron accumulation are also important features of the PD neuropathology (74, 75). As observed in PD brain (76), SNc and striatum of aged c-rel*−*/*−* mice showed marked signs of microglia activation with increased number, swollen cell bodies, and thick processes of CD11b-positive cells. Both SNc and striatum of aged c-rel*−*/*−* mice displayed elevated levels of iron and increase of the iron transporter DMT1 that, as mentioned above, is a transcriptional target of aberrantly acetylated RelA. Preliminary investigation on NF-κB in striatal extracts of 18-month-old c-rel*−*/*−* mice confirmed, indeed, the presence of aberrantly acetylated RelA as reported in **Figure 2**. The immunoprecipitated RelA showed reduced level of general acetylation associated with increased site-specific acetylation at the K310 residue. These changes occurred without any significant variation in the cellular amount of RelA. This finding strongly

**(A)** Representative picture of the immunoprecipitation analysis of RelA acetylation in total proteins of caudatus putamen. RelA acetylation at K310 residue increased, while total RelA acetylation was reduced in striatal total extracts of 18-month-old c-rel*−*/*−* mice. No significant change was detected in the total RelA content. **(B)** Values from densitometry analysis of immunoblots are expressed as a percentage of the wt value. The signal given by IgG(H) is used as a control for the quality of the immunoprecipitation. Bars depict the mean *±* SEM (*n* = 4 animals per group), \**p <* 0.01 versus wt value.

suggests that a misbalance between c-Rel and RelA can evolve during aging in c-rel*−*/*−* mice to produce changes in RelA acetylation, microglia activation, and neuronal apoptosis.

Because inflammatory and neurotoxic activation of microglia have been also reported to be triggered by acetyl-RelA (K310) (77), it is feasible that microglia activation participate to the neurodegenerative process in c-rel*−*/*−* mice. The extensive analysis of the neuroinflammatory profile of c-rel*−*/*−* mice along with disease progression will reveal the exact entity of this inflammatory process and the specific participation of innate and adaptive immunity.

The neurochemical changes observed in aged c-rel*−*/*−* mice were also accompanied by the onset of motor deficits. A significant impairment in spontaneous motor activity was evident in c-rel*−*/*−* mice at 18 months, but not in younger mice as previously shown (17, 18). Indeed, either monitored for 1 h or six consecutive days to avoid stress-related bias, 18-month-old c-rel*−*/*−* mice displayed a lower locomotor activity. Furthermore, the gaiting analysis supported the presence of a locomotor dysfunction related to bradykinesia and rigidity. Noteworthy, the treatment with -DOPA plus benserazide, a cocktail that is considered the gold standard for PD therapy, totally reversed the locomotor deficits and normalized most of the gaiting parameters.

Despite these findings, how the constitutive c-Rel deficiency can specifically affect DA neurons of SNc is still an open question (10). The selective vulnerability of SNc neurons in PD has been attributed to the peculiar "energy-demanding" physiology of these cells (78), which display enormous axonal field and impressive number of synapses for each axon (79). Moreover, during their pacemaking activity, SNc DA neurons, but not the ventral tegmental area neurons, generate autonomous action potentials by unusual engaging of L-type Ca2+ channels, which require subsequent activation of ATP-dependent Ca2+ pumps to maintain Ca2+ homeostasis (80). The energy production by mitochondria and endoplasmic reticulum in SNc DA neurons associates with the generation of large amounts of ROS that are constantly neutralized by anti-oxidant systems, including SODs catalases, glutathione peroxidase, and UCP4 and UCP5 (81). It can be inferred that in the absence of c-Rel a reduced expression of UCP4 (31) and MnSOD (7, 29) might enhance ROS accumulation during aging in SNc neurons (82), and synergize with reduced expression of antiapoptotic Bcl-xL (9, 30) to affect neuronal resilience. Also, it is conceivable that mitochondria impairment associated with c-Rel deficiency may switch the acetylation state of RelA during aging to elevate Bim, DMT1, and iron (75) as well as the intracellular levels of α-synuclein (83). In turn, these events lead to α-synuclein aggregation (83), microglia activation, and neuronal damage (84). All together, these findings point to a role of c-Rel in the regulation of SNc susceptibility to aging.

Finally, latest results (unpublished results) indicate that at a premotor phase (7–12 months) when no loss of SNc DA neurons is evident yet, c-rel*−*/*−* mice display olfactory deficits, gut dysfunctions, and reduced DAT immunoreactivity in the striatum. This disease progression mimics the pathological and clinical progression observed in PD patients that at premotor stage of the disease show constipation, hyposmia (85), and reduced DAT imaging by PET or SPECT scan. These findings further strengthen the notion that c-rel*−*/*−* mice represent an innovative disease model suitable both for studies aimed at dissecting the mechanisms of PD onset and to test novel therapeutic approaches for intervention at the premotor stages of the disorder.

# **Conclusion**

Although NF-κB factors are transcriptional regulators of inflammation and apoptosis, their relevance in aging-related neurodegeneration is still underestimated.

Activation of RelA has been proposed to lead the systemic aging process in mice (86, 87), being negligible in the hypothalamus of young mice and progressively increasing, earlier in microglia and later in neuronal cells, as the mice become older (12). The genetic depletion of one allele of RelA attenuated the behavioral signs of neurodegeneration and extended the healthspan in a progeroid mouse model (13).

These stimulating results could be reread in the light of recent evidence showing that protective versus harmful p50/RelA activation strongly depends on the acetylation state of RelA. Activation of RelA subunit displaying reduced grade of general acetylation, but site-specific acetylation of K310, triggers apoptotic gene expression in brain ischemia. In the absence of such a mismatch, the activation of RelA is neuroprotective, as observed in preconditioning ischemia.

Whether a dysregulation of the RelA acetylation state is also involved in normal aging, or just in pathological aging, remains to be established. What seems promising is that aberrant RelA acetylation, more than RelA nuclear translocation,

# **References**

can be a suitable target. It is corrected by the synergistic association of HDAC inhibitors and resveratrol to produce neuroprotection.

While evidence suggests that RelA activation marks physiological elderly in mice, we show that the deficiency of c-Rel leads to a parkinsonian phenotype with aging (10). The degeneration of DA neurons in the SNc, the microglia activation and the αsynuclein pathology are associated with development of an - DOPA-responsive parkinsonism. Intriguingly, in the basal ganglia of aged c-Rel deficient mice, but not in aged matched controls, the acetylation state of RelA was reminding the one observed in lethal ischemia.

This body of evidence supports the premise that the balance between c-Rel- and RelA-mediated transcription may be at the crossroad between normal and pathological aging of the mammal brain. A defect of c-Rel activity, associated with higher RelA activation, reduces SNc resilience to aging hereby leading to a late-onset form of parkinsonism. Validation of the impact of c-Rel activity in PD onset and progression is now the challenge of ongoing studies.

# **Acknowledgments**

This work was supported by Ex 60% 2008–2011 from the University of Brescia; NEDD Project (CUP H81J09002660007), Regione Lombardia, Italy; Ricerca Finalizzata Ministero della Salute Italy RF-2010-2315142; Marie Curie Actions – ITN nEUROinflammation, FP7 Project No. 607962 Fondazione CARIPLO 2014-0769, Italy.

mouse in response to ischemia-reperfusion injury. *Stroke* (2006) **37**(6):1533–8. doi:10.1161/01.STR.0000221708.17159.64

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Guest Associate Editor Giuseppe Pignataro declares that, despite having collaborated previously with the authors Annamaria Lanzillotta, Caterina Branca, Pier Franco Spano and Marina Pizzi, the review process was handled objectively and no conflict of interest exists.

*Copyright © 2015 Lanzillotta, Porrini, Bellucci, Benarese, Branca, Parrella, Spano and Pizzi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Dynamic changes in DNA methylation in ischemic tolerance**

*Robert Meller 1 \*, Andrea Pearson1 and Roger P. Simon1,2*

*1 Translational Stroke Program, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA, USA, 2 Grady Memorial Hospital, Atlanta, GA, USA*

Epigenetic mediators of gene expression are hypothesized to regulate transcriptomic responses to preconditioning ischemia and ischemic tolerance. Here, we utilized a methyl-DNA enrichment protocol and sequencing (ChIP-seq) to identify patterns of DNA methylation in an established model of ischemic tolerance in neuronal cultures (oxygen and glucose deprivation: OGD). We observed an overall decrease in global DNA methylation at 4 h following preconditioning ischemia (30 min OGD), harmful ischemia (120 min OGD), and in ischemic tolerant neuronal cultures (30 min OGD, 24 h recovery, 120 min OGD). We detected a smaller cohort of hypermethylated regions following ischemic conditions, which were further analyzed revealing differential chromosomal localization of methylation, and a differential concentration of methylation on genomic regions. Together, these data show that the temporal profiles of DNA methylation with respect to chromatin hyperand hypo-methylation following various ischemic conditions are highly dynamic, and may reveal novel targets for neuroprotection.

#### *Edited by:*

*Giuseppe Pignataro, University of Naples Federico II, Italy*

#### *Reviewed by:*

*Zhihui Yang, University of Florida, USA Agnese Secondo, University of Naples Federico II, Italy*

#### *\*Correspondence:*

*Robert Meller, Neuroscience Institute, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30310, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology*

> *Received: 23 February 2015 Accepted: 24 April 2015 Published: 15 May 2015*

#### *Citation:*

*Meller R, Pearson A and Simon RP (2015) Dynamic changes in DNA methylation in ischemic tolerance. Front. Neurol. 6:102. doi: 10.3389/fneur.2015.00102* **Keywords: DNA methylation, ischemia, preconditioning, stroke, ischemic tolerance, ChIP-seq**

# **Introduction**

Pharmacological agents for neuroprotection have been disappointing in clinical trials. An understanding of the basic mechanisms of endogenous neuroprotection in brain, those induced by preconditioning ischemia and resulting in a tolerant phenotype, may yield novel approaches to reduce brain injury following ischemia (1, 2). We have previously described broad-based transcriptional suppression as operative in ischemic tolerance (3), and have discovered protein effectors of tolerance: upregulation of gene silencing polycomb proteins in tolerant brain (4). Polycomb protein's mechanism of action is via epigenetic chromatin binding, highlighting a putative biologic mechanism with neuroprotective potential.

It has been proposed that a reduction in DNA methylation will confer protection against brain ischemia. Global DNA methylation increases in the brain following harmful focal ischemia, as measured by [3H]-methyl incorporation into DNA (7), but does not change the expression of DNMT1 or 3 (DNA methyltransferase, DNMT). However, the chromatin sequences that are methylated by ischemia were not identified by Endres (7). Blocking the enzyme responsible for DNA methylation, DNMT, using 5*′* azacytidine inhibits ischemic cell death (7). Heterozygous, but not homozygous knockouts of DNA methyltransferase1 also show a reduction in brain infarction following modeled ischemia (8). Therefore, reducing DNA methylation may protect the brain from subsequent harmful ischemia.

An increase in DNA methylation following ischemia would be consistent with a recent study which showed that MECP2 (methyl CpG binding protein 2), a transcription repressor, is increased following brief ischemia (9). In addition, the DNA methylating agent methylazoxymethanol (MAM) blocks ischemic tolerance induced neuroprotection (10). In that study, while the effect was attributed to a blockade in progenitor cell proliferation, the ability of MAM to hypermethylate DNA such that preconditioning-induced changes in DNA methylation are blocked, cannot be ruled out. While it is unclear which genes are methylated in response to ischemia, ischemia can affect DNA methyltransferase activity in brain, and a reduction in DNA methylation may play a role in mediating the protective effects of ischemic tolerance.

The role of DNA methylation was recently studied in an analogous model of ischemic tolerance that of tolerance to seizureinduced brain injury (11). Following preconditioning seizures, brain injury following status epilepticus induced by an intraamygdaloidal injection of kainic acid is reduced, specifically in the CA3 subfield of the hippocampus (12–14). Similar to ischemic tolerance, a reduction in gene expression has been reported in seizure tolerant brain, suggesting gene repression mediates tolerance to both seizure and ischemic brain injury (13). In the seizure tolerance study, DNA methylation patterns were profiled at 8 h post seizure, and revealed a dynamic pattern whereby both an increase and decrease in methylation were observed (11).

Thus, the role of DNA methylation in ischemic tolerance is not clear. Here, we offer an in-depth analysis of DNA methylation in a cell culture model of ischemic preconditioning-induced ischemic tolerance (15).

# **Materials and Methods**

# **Cell Culture and Ischemic Tolerance Modeling**

All animal use was approved by the Morehouse School of Medicine Institutional Animal Care and Use Committee. Primary neuronal cultures were prepared for mixed sex litters of rats (1 day post birth). Animals were euthanized with isoflurane, and cortices dissected in an ice cold buffer (dissociation media). Tissue was incubated with papain (37°C: 10 min) and triturated. Cells were seeded out a 5 million cells/6 cm dish. Cells were grown in a mixture of B27 (3.5%) and Neurocult (1.5%) supplemented NeuroBasal A media. Cells were used at 14 days *in vitro* (DIV).

An *in vitro* equivalent of ischemic tolerance is used, whereby primary neuronal cultures are subjected to oxygen and glucose deprivation (OGD) modeled ischemia (16–18). Cells are washed with phosphate buffered saline, supplemented with Mg Cl2 and Ca Cl2, and then incubated for 30 or 120 min in a Thermofisher anaerobic chamber. Cells were recovered in neurobasal A media. For modeling ischemic tolerance, we precondition cells with 30 min OGD, and injurious ischemia (120 min) is applied 24 h later. Cells were harvested 4 h following the final ischemic challenge.

# **DNA Preparation and Methylation Enrichment**

Methylated DNA was enriched from cell lysates (*n* = 2/condition) using the Methylminer assay (Active Motif). DNA was extracted from frozen cell pellets using the Genelute DNA Kit (Sigma). DNA was quantified and 2.0 μg was sonicated in 200 μl Tris EDTA buffer (1 *×* 30% 10 s, 2 *×* 20% 10 s, 4°C). Sheared DNA (2000 ng) was used as starting material for the pull down reaction according to manufacturer's protocol. Following pull down, the DNA was size selected on a 1% agarose gel [200–500 base pair (bp) fragments] and extracted using a PCR (polymerase chain reaction) clean-up kit (Qiagen).

### **DNA Library Preparation**

Samples were end-polished and ligated to barcode adapters using T4 Kinase. Libraries were amplified using Platinum taq for 12 cycles of PCR, size selected on a 2% agarose gel, and then subjected to an additional four cycles of PCR. Libraries were visualized on an agarose gel (see **Figure 1A**), excised, and cleaned up to remove primers (Qiagen). Samples were then subjected to qPCR to quantify the library against a known standard. Emulsion PCR was used to clone the libraries onto sequencing beads. The reaction was seeded at 1.0 pmol of equimolar library (all eight samples at the same concentration). A WFA run determined the final concentration of beads for deposition on the sequencing slide to obtain approximately 700 million beads deposited on the slide. Libraries were sequenced on an Applied Biosystems SOLiD 4 DNA sequencer using a F35 single end read. Data were stored as csfasta files, and uploaded to a penguin cluster running LifescopeTM for alignment to the RN5 genome (rat), using the RefSeq annotation guide (October 2014).

# **Data Analysis**

Data were transferred to a Dell Dimension Desktop computer running Partek Genomic Studio v 6.6 for analysis. Data were analyzed using the Partek ChIP and the NGS Methylation workflows. Output bam files were combined according to experimental conditions for all analysis. Data were subjected to cross strand analysis to identify the fragment window size for genomic analysis (**Figure 1C**). Data were aligned to the rn5 genome, using the control sample as the reference, and a window bin size of 200 bp. Only fragments that show enrichment with a *p <* 0.001 false detection rate (FDR) were considered significant and identified in a "peaks file." These data were used for subsequent analysis (available upon request).

# **Results**

# **Ischemic Stimuli Result in a Reduction in Global DNA Methylation**

Methylation profiles of ischemia treated neuronal cell cultures were identified using methylated chromatin enrichment followed by DNA sequencing (ChIP-seq). We analyzed four treatment groups: sham-treated cells (Control: Cont), cells subjected to 30 min OGD (Preconditioning: PC) and recovered for 4 h, cell subjected to 120 min ischemia (Injurious Ischemia: Isch), and neurons subjected to 30 min OGD and recovered 24 h prior to harmful ischemia (Tolerance: PC + Isch). These conditions have been established in multiple studies as showing ischemic tolerance (3, 16, 18). We focused on a time point of 4 h following the last ischemic treatment to identify dynamic changes in methylation that may be responsible for subsequent gene expression changes.

Quantitative analysis of assembled libraries on an agarose gel suggests a global decrease in DNA methylation, detected using the Methylminer assay. Interestingly, methylation was reduced following all ischemia treatment protocols (**Figures 1A,B**). Libraries were assembled and DNA concentrations were normalized prior to sequencing. The sequenced data were aligned to the rn5 reference genome; there were similar numbers of alignments in each experimental group (mean of 20.2 million reads *±* 1.8 million

reads; **Figure 1C**). The equivalent number of aligned reads is due to the concentration normalization step in the sequencing protocol. Therefore, all subsequent analysis reveals differences of methylation enrichment patterns rather than absolute DNA methylation levels.

In order to compare datasets, we combined the reads from each experimental condition (Cont, PC, Isch, PC + Isch), resulting in 30–40 million aligned reads per condition. Global methylation status was visualized with a Hilbert graph (19). Intensity of methylation is represented by red, and weaker intensity is represented as blue. Similar to previous published studies, the visualization of DNA methylation patterns showed a diffuse pattern of reads across the genome (**Figure 1D**). However, it is of note that the intensity of reads is lower globally across the genomes of the PC, Ischemia, and PC + Ischemia treated cells, consistent with the library analysis (**Figure 1A**). Of note, we also observed a lower global representation of methylation on the X chromosome (**Figure 1E**).

size for peak analysis. The PC and ischemia curves overlap (Cont: blue, PC: green, Isch: red, and PC + Isch: black). **(B)** Quantification of number of peaks in each treatment condition, and the number of unique peaks in each condition denoted by an asterisk. Data from the two sets were combined.

peaks and per Mbp of chromosome to reveal selective enrichment of chromosome patterns of DNA methylation. Note high relative methylation of mitochondria (discontinuous axis).

A peak analysis was performed on the DNA-seq data to identify clusters of methylation and relative differences in methylation compared to control. The window for the fragment size for analysis was determined by performing a cross strand analysis (20) (**Figure 2A**). Samples showed an approximate peak of around 150 bp (arrow **Figure 2A**). Reads were analyzed using a 150 bp fragment window, and using the control samples as a reference (to compare DNA methylation to control). The genome was divided into 100 bp windows, which could be combined. Data were fit to a negative binominal model with a peak cutoff false detection rate (FDR) of *p <* 0.001. From this analysis, 5035 peaks were identified in each of the four conditions. When we plot the number of methylation peaks (**Figure 2B**), we observed that the number of peaks decreases in the PC, Ischemia, and PC + Ischemia treated cells. The number of peaks unique to each condition are also plotted, and these have a similar distribution (peaks unique to each condition are marked with an asterisk; **Figure 2C**). Interestingly, although global methylation is reduced in ischemia-treated cells compared to PC-treated cells (**Figure 1B**), the number of hyper methylated regions is the same (**Figure 2B**). Our interpretation of this result is that global levels of DNA methylation are reduced 4 h following harmful ischemia, but individual genes may show a strong increase in methylation, which we further investigated (below).

# **Dynamic Changes in Methylation Following Ischemia Show Chromosomal and Functional Bias**

In order to identify genes whose methylation increased following ischemia treatments, we subjected the data to a one-tailed negative binominal test and selected a cut off FDR value of 0.05. Compared to the control sample, 618 regions show significant enrichment, which we interpreted as increased methylation. There were more enriched regions in the ischemia group (345) compared to those treated with preconditioning only (187), or in tolerant samples (65) (**Figure 2D**).

We separated the differentially methylated peaks by treatment, and identified which genomic feature they were close to and which chromosome they align with. Analysis of the enriched methylation peak distribution revealed an increase in chromosome 2, 12, and 13 methylation in the ischemia-treated cells (**Figure 2C**). In order to assess relative chromosomal distribution, we normalized the number of peaks to the total number of peaks/condition, and then scaled to the size of the chromosome (**Figure 2D**). Global methylation was reduced following PC + Ischemia/tolerance (**Figure 1B**); it is of note that chromosome 1, 7, and 17 showed relative enhancements in hypermethylation compared to control. Following PC, methylation of chromosome 6, 9, 16, and 20 showed enhanced hypermethylation compared to control. There was no chromosome selective enrichment of hypermethylated regions in ischemia-treated cells; rather, hypermethylated chromosomes were common preconditioned cells (chromosome 2 and chrX), or preconditioned cells subjected to harmful ischemia (chromosome 3, 12, and 13) (**Figure 2D**). This suggests that different chromosomes show predominant methylation in response to various ischemic treatments, which may result in differential activation of gene expression/repression in response to these stimuli.

The lists of significantly regulated genes were then analyzed, according to which genomic feature they correspond with using the RefSeq Transcripts Annotation guide (24 October, 2014) (**Figure 3A**). Promoters were defined as 5000-0 bases downstream of the transcription start site. Data from multiple introns and exons (CDS) were combined into one category. The most notable feature was the very high representation of intergenic regions in this data set; yet, only 1% of the genome encodes genes. It is not clear whether these intergenic associated regions of methylation are associated with non-transcribed DNA or novel transcripts of RNA, which are as yet unannotated (or not in the refSeq guide). Ischemia-treated and tolerant cells appear to have similar concordance with a control (total) distribution of methylation across genomic features. In contrast, following preconditioning ischemia, there was a relative increase in PC-mediated exon and intron-associated methylation, and a decrease in intergenic methylation (**Figures 3A,B**).

Finally, we investigated the location of reads relative to the transcription start site (TSS). This analysis is independent of the gene name; so, it only considers the relative location of peaks to the closest TSS. All samples had a similar profile in that we saw a relatively even distribution before and after the TSS (**Figure 3B**). A notable exception is at the actual TSS where the depth of reads is higher. When we expand this region (**Figure 3C**), we observed that control, PC, and ischemia treated samples showed a clustering around the TSS; in contrast, the largest peak from the tolerant cells was approximately 150 bp downstream of the TSS. Whether this was due to the loss of global methylation in these samples or a more selective methylation mark close to the TSS is not yet clear. Similarly, samples from PC and Ischemia treated cells also show a peak, but closer to 100 bp downstream of the TSS. The significance of these marks on gene expression awaits further investigation.

# **Pathway Analysis of Methylated Regions**

For each treatment group, we created lists of genes closest to the methylation peaks, and genes unique to each treatment group. We performed a pathway analysis on the gene lists associated with methylated regions. Fisher's exact test was performed, and pathways were restricted to have at least two genes present. From this analysis, we first looked at enrichment scores and enrichment *p*-values, with a cut off of *p <* 0.05. (**Table 1A** depicts the pathways associated with the methylated genes in control, and 1B unique to control). In control samples, 13/19 of the pathways were associated with immune and inflammatory responses, or were identified as having genes associated with the immune system. Unique to control, we observe 19 pathways showing enrichment, again showing a high number of immune system associated pathways (**Table 1B**).

Following preconditioning ischemia, 17 pathways are identified, 10 of which are immune system, but 2 are associated with serotonin (**Table 2**). In the unique PC gene list, two-third of the pathways identified with synaptic pathways rather than the immune system. Following harmful ischemia (**Tables 3A,B**), 15 pathways showed enrichment, of which 13 are immune function associated. When we look at pathways unique to ischemia, three pathways associated with the synapse and receptor ligand interactions are identified. In ischemic tolerance, we do not observe any significant pathways identified, but one pathway, insulin secretion, was identified in the unique tolerance sample (**Table 4**). The low number of pathways identified in the tolerant sample is due to the low number of methylation associated peaks identified in these samples.

# **Discussion**

Methylation of DNA is an important mechanism by which epigenetic modulation of gene expression occurs. Here, we show that both protection inducing ischemia (preconditioning) and harmful ischemia reduce global DNA methylation. The decrease in global methylation was more pronounced in ischemic tolerant cells. While global methylation was reduced, we observe an increase in methylation in chromosome 2, 12, and 13 in ischemia-treated cells and chromosome 1, 7, and 17 in tolerant cells, suggesting selective enhancement/targeting of methylation at these chromosomes.

While the distribution of methylation across ischemic and tolerant genomic regions was similar in incidence, in the preconditioned cells an exon associated methylation induction and attenuation in intergenic methylation were noted. A clustering of differential methylation at transcriptional start sites was observed in all cell treatment groups.

Similar global changes in methylation are reported in brain following injurious status epilepticus when compared to changes seen after induction of seizure tolerance (11). The hypothesis in both our ischemia studies and those of seizure was that the activation of neuroprotective mechanisms via previously described genomic reprograming and transcriptional suppression might occur via epigenetic processes, specifically by DNA methylation. However, in both our study of ischemia and the seizure study, a profound global hypomethylation event was observed in response to cell stress (ischemia or seizures). While this contrasts with studies showing increased methylation following harmful ischemia (7), it should be noted that the present tolerance and seizure studies used shorter recovery times following the stress 4 h (present study) and 8 h (11). It should also be noted that 4 h following preconditioning ischemia and preconditioning seizures, we have noted larger changes in gene expression compared to 24 h post preconditioning [(21) and Meller, unpublished].

Global demethylation was more pronounced in the ischemic tolerant group (cells subjected to preconditioning and harmful ischemia) compared to the ischemia or PC only-treated cells. It is not clear whether the reduction in methylation is due to the **TABLE 1 | (A) List of pathways identified from closest gene to methylation peaks in control MeDNA sample; (B) list of pathways identified from closest gene to methylation peaks unique to control MeDNA sample**.

repeated exposure of the tissue to ischemic stress, resulting in an additive effect, or whether it is unique to the tolerance phenomenon. Hypermethylation was also reduced in this group compared to the other ischemia treatments. Since fewer "enriched" hypermethylation peaks were identified in the tolerance group, our mapping software only found one regulated pathway (insulin secretion). A reduction in hypermethylation in ischemic tolerance was, perhaps, unexpected given the established observations of gene silencing in ischemic tolerance. Indeed, on first appearance our data appear to contradict the hypothesized dogma that ischemic tolerance is associated with gene silencing (3, 4). However, care should be taken in interpreting these data sets together in this way, given that the current ischemic tolerance study identified chromatin methylation status 4 h following the final harmful insult, whereas gene expression and protein levels were reported 24 h following the final insult (3, 4). These discrepancies clearly support the need for further detailing the temporal profiles of both gene expression and chromatin methylation in response to such stimuli.

Most of the differential methylation is intergenic, which could associate with either non-transcribed DNA regions, or currently unannotated RNA species in the genome. RNA sequencing based transcriptome analysis shows approximately 30% of RNA is from such unannotated regions (22). It is interesting that there is a reduction in methylation in these regions of PC treated cells. When we remove the intergenic reads (**Figure 3B**), we observed that following PC there is a relative increase in methylation in coding regions compared to harmful ischemia in which methylation was focused more in intergenic regions. Whether this suggests changes in structural organization of chromatin or noncoding RNAs remains to be determined. In tolerant cells, we observed a reduction in coding region associated with methylated peaks. This may be due to the lower number of reads in this sample set, or a genuine decrease in exon associated methylation. **TABLE 2 | (A) List of pathways identified from closest gene to methylation peaks in PC MeDNA sample; (B) list of pathways identified from closest gene to methylation peaks unique to PC MeDNA sample**.

**TABLE 3 | (A) List of pathways identified from closest gene to methylation peaks in ischemia MeDNA sample; (B) List of pathways identified from closest gene to methylation peaks unique to ischemia MeDNA sample**.

#### **TABLE 4 | List of Pathways identified from closest gene to methylation peaks unique to tolerance MeDNA sample**.

Meller et al. DNA hypomethylation following ischemia

Additionally, methylation of intronic regions was increased following PC. This may be due to non-coding RNAs associated with intronic regions, or may represent regulation of alternative splicing of gene transcripts, which may occur following preconditioning (Meller, unpublished observation) or ischemia (23).

Following ischemia treatments, we observed a chromosomal difference in patterns of enhanced methylation compared to control cells. This suggests that chromosomal specific patterns of chromatin methylation may be regulated following various ischemic stimuli. How specific chromosomes are targeted for enhanced methylation is not clear, but studies of methylation following seizures support differential chromosomal location of methylation events following stress (11). Our analysis focused on specific increases in methylation clusters, because we observed global decreases in methylation events. Chromosomal specific gene expression effects of excitotoxicity have been reported previously (24). Chromosomal bias may be associated with movement of chromosomes into active nuclear territories (25). For example, chromosome X movement has been described following epilepsy (26). Changes in nuclear structure and epigenetic marks have been reported in response to synaptic and excitotoxic signaling (27).

Our study may upon first analysis appears to contradict the study of Endres; however, it should be noted that very different methodologies were utilized in these studies. In the Endres study, the incorporation of radiolabeled methyl groups into genomic DNA was measured. As such increased methylation may be due to dynamic DNA methylation or DNA methylation following DNA damage and repair, which has been reported following ischemia (28, 29). We also observed a global decrease in methylation following harmful ischemia, and DNA damage would be expected following harmful ischemia, hence re-methylation may occur. DNA methylation blockers are neuroprotective against ischemia (7). The agents were administered 10 min prior to harmful ischemia. However, the temporal profile of protection afforded by such blockade is not clear. In our study, we observed a reduction in global DNA methylation in tolerant neurons, suggesting that preventing the increased specific methylation observed following harmful ischemia could be protective.

In contrast, our study correlates with the observations of Miller-Delaney that DNA hypomethylation is associated with neuroprotection. In the current study, a methyl DNA binding

# **References**

protein related to the methyl binding domain of MeCP2 was used to enrich chromatin, in contrast to an anti-Me-cytosine antibody enrichment procedure (11). Increased MeCP2 has been reported in brain 24 h following preconditioning ischemia due to the loss in the levels of miR-132 (9). Thus, some of the differences in individual gene/chromatin region identification may be due to methods of enrichment. We also utilized DNA sequencing of the whole rat genome vs. mouse promoter arrays (11), which may also reveal differences between studies. Furthermore, sequencing libraries were normalized to approximate equimolar concentrations, prior to seeding the cloning PCR. The effect of this is to ensure even representation of the libraries in the sequencing reactions. However, as a result, some of the global decreases in methylation become less apparent. This was in part controlled for by comparing the methylation to the control sample, thereby revealing the dynamic changes in methylation. Clearly, more analysis of such studies will reveal additional information, especially when more advanced analysis methodologies and tools become available to incorporate such aspects of sequencing.

In summary, we performed a DNA methylation study using next generation sequencing which revealed dynamic changes in DNA methylation following preconditioning ischemia and in ischemic tolerance. Our study showed that 4 h following preconditioning ischemia, harmful ischemia, and in tolerant cells, DNA hypomethylation predominates as the response. However, selective and significant increases in hypermethylation events can also be observed, especially in response to harmful ischemia. The consequence of these events upon transcription awaits definition, but this study suggests that the temporal profile of epigenetic regulation of gene expression events needs further study to improve our understanding of this critical transcription control mechanism, and the identification of novel targets for neuroprotection from stroke induced brain injury.

# **Acknowledgments**

RM was supported by NS59588. Institutional support was provided by U54NS060659 and U54NS0839321. This publication is supported by the Research Centers in Minority Institutions (RCMI) Grant Number 8G12MD007602 from the National Institute of Minority Health and Health Disparities (NIMHD).

epileptic tolerance. *J Neurosci* (2012) **32**:1577–88. doi:10.1523/JNEUROSCI. 5180-11.2012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Meller, Pearson and Simon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Extending injury- and disease-resistant CNS phenotypes by repetitive epigenetic conditioning

## **Jeffrey M. Gidday 1,2,3\***

1 Department of Neurosurgery, Washington University School of Medicine, St. Louis, MO, USA

2 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

3 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA

#### **Edited by:**

Giuseppe Pignataro, University of Naples Federico II, Italy

#### **Reviewed by:**

Luigi Formisano, University of Sannio, Italy Roger Simon, Morehouse School of Medicine, USA

#### **\*Correspondence:**

Jeffrey M. Gidday, Department of Neurosurgery, Washington University School of Medicine, Box 8057, St. Louis, MO 63110, USA e-mail<EMAIL_ADDRESS>
Significant reductions in the extent of acute injury in the CNS can be achieved by exposure to different preconditioning stimuli, but the duration of the induced protective phenotype is typically short-lasting, and thus is deemed as limiting its clinical applicability. Extending the period over which such adaptive epigenetic changes persist – in effect, expanding conditioning's "therapeutic window" – would significantly broaden the potential applications of such a treatment approach in patients. The frequency of the conditioning stimulus may hold the key. While transient (1–3 days) protection against CNS ischemic injury is well established preclinically following a single preconditioning stimulus, repetitively presenting preconditioning stimuli extends the duration of ischemic tolerance by many weeks. Moreover, repetitive intermittent postconditioning enhances post-ischemic recovery metrics and improves long-term survival. Intermittent conditioning is also efficacious for preventing or delaying injury in preclinical models of chronic neurodegenerative disease, and for promoting long-lasting functional improvements in a number of other pathologies as well. Although the detailed mechanisms underlying these protracted kinds of neuroplasticity remain largely unstudied, accumulating empirical evidence supports the contention that all of these adaptive phenotypes are epigenetically mediated. Going forward, additional preclinical demonstrations of the ability to induce sustained beneficial phenotypes that reduce the burden of acute and chronic neurodegeneration, and experimental interrogations of the regulatory constructs responsible for these epigenetic responses, will accelerate the identification of not only efficacious but also practical, adaptive epigenetics-based treatments for individuals with neurological disease.

**Keywords: stroke, neuroprotection, preconditioning, postconditioning, epigenetics, retina**

### **STATE OF THE ART**

It is now widely established that organisms, tissues, and cells respond to sublethal stressors by a transient augmentation in their innate capacity to resist injury. That low doses of a harmful stimulus can promote beneficial responses forms the basis of hormesis theory (1), but only relatively recently have the conceptual and mechanistic connections between conditioning-induced tolerance and the hormetic response been recognized (2–5). In any event, seeking to leverage conditioning- or hormesis-based adaptive responses in patients suffering from acute ischemia, we remain somewhat stymied by several features of our laboratory models that seem to preclude easy translation to the clinic.

Significant progress has been made in preclinical models that advance our understanding of the molecular and genetic mechanisms underlying ischemic tolerance in the CNS (6–9). Many unique subcategories of this general phenomenon have been identified and subject to experimental scrutiny (8). For example, we recognize two basic phases or therapeutic windows for conditioning-induced protection: an early "rapid preconditioning" phase that is manifested nearly immediately after the adaptive stimulus is presented, which is largely dependent on rapid post-translational protein modifications, and a delayed preconditioning phase that takes several hours, sometimes days, to fully develop, resulting from changes in gene expression. We know that improved outcomes can result if the conditioning stimulus is applied to another tissue ("remote conditioning"), although its neural/humoral mechanism remains elusive. We have also learned that, even when initiated after the injurious event ("postconditioning"), conditioning stimuli can still afford protection. However, in all of these conditioning paradigms, we have defaulted to the assumption that the duration of the resultant injury-tolerant state requires a defined period of time to become established, and, more importantly, that it cannot be sustained indefinitely. This latter supposition has hindered, directly or indirectly, the clinical applicability of this phenomenon, as physicians face the daunting challenge of how best to pre- or post-condition patients at a welldefined higher risk for an ischemic event, let alone post-condition those stroke patients that present in the absence of recognizable risk factors. Leveraging such innate adaptive responses to treat chronic neurodegenerative disease seems even more unlikely, if not impossible.

Against this backdrop, I explore herein the contention that, as a result of epigenetics-based changes in gene expression, the duration of injury resistance following a stress stimulus can be

increased quite significantly based on specific manipulations of the "dose" of the conditioning stimulus. Further interrogation of how best to establish protracted therapeutic windows of innate neuroprotection will provide some of the most valuable opportunities for multiplying the clinical potential of conditioning to prevent and treat both acute and chronic neurological disease.

### **REDEFINING STIMULUS-RESPONSE**

Throughout most of its history at both the bench and in the clinic, the conditioning/tolerance field has evolved based on a fundamental, implicit definition of the very phenomenon we have come to understand: A transient, but robust, reduction in injury from acute ischemia can be realized if the tissue is preconditioned "X" hours prior to the lethal ischemic insult. For the aforementioned delayed phase of conditioning, the words "transient" and "X" in this definition have been understood to be ~8–48 h, essentially bookending the "therapeutic window" for treatment. Similarly, for postconditioning, preclinical protocols typically involve conditioning shortly after the ischemic event, as well as performing relatively early assessments of efficacy based largely on lesion quantification. Few studies have examined whether the duration of tolerance resulting from pre- or post-conditioning can be extended secondary to manipulating the "dose" of the conditioning treatment, or the related possibility that repetitive postconditioning during recovery can not only reduce injury but concomitantly enhance one or more long-term stroke recovery metrics; both of these advances would portend powerful clinical implications. In dose–response parlance, if the goal is to invoke a protracted response or prolonged period of tolerance to a given dose of conditioning, and if the given period of tolerance currently appears time-limited, then a closer examination of the conditioning dose is required to achieve this desired response. This has not really happened in any systematic sense; rather, we were quick to adopt the general assumption that the only type of tolerance is one defined by a short therapeutic window.

Every stimulus can be defined by at least three parameters: magnitude, duration, and frequency. Until the advent of postconditioning by instituting cycles of brief ischemia during early reperfusion, the vast majority of preconditioning paradigms for stroke have historically involved a single stimulus (i.e., frequency = 1) of a specific duration and magnitude that set into motion tolerancesignaling cascades; in the field of myocardial ischemia, repetitive preconditioning treatments have been explored (10), but not for the purpose of identifying a treatment that extends the duration of tolerance. Often overlooked is the fact that, like every stimulus, every response can also be defined by at least two of the same three parameters: its robustness and its duration. However, the preconditioning field settled quickly on a magnitude-focused, binary definition of tolerance as protection/no protection measured within ~24–72 h of conditioning; little attention has been focused on whether the duration of a given protective response – again, what is essentially the therapeutic window for this treatment – could be extended. The most obvious way to do so involves changes in stimulus frequency.

In fact, stimulus frequency is critical not only for determining the duration of the phenotypic change induced but also for its cumulative ability to promote an adaptive response, in contrast to the potential impotence of a single exposure or the potential injury resulting from multiple exposures combined as one or administered too closely together. Indeed, evidence emerging not only in the field of conditioning but across other dimensions of biology as well indicates that the frequency of the presented stimulus may be the key dose feature with respect to generating a beneficial, adaptive response instead of a harmful, maladaptive response (or no response at all). Exercise represents one example of benefit-inducing repetitive conditioning (11); moreover, recent clinical studies support the contention that increasing the number of short duration/low intensity bouts of exercise may provide more "advantages" to the cell, tissue, or organism than fewer exposures that are longer and/or more severe (and thus may afford no adaptive benefit or even cause injury) (12). As another example, repetitive intermittent hypoxia, initiated after incomplete spinal cord injury, promotes persistent functional recovery of respiratory and non-respiratory motor systems, as manifested by protracted improvements in gait performance in both rodents (13) and humans (14). In the field of psychology, the enhanced neuronal plasticity that promotes resilience – instead of vulnerability – to a number of different stress paradigms is becoming recognized as uniquely dependent on the frequency of exposure to the stressor (15–21). The long-term effects of intermittent fasting, and meal frequency and timing, on health and disease outcomes (22) represent additional facets of this same concept. Finally, repeated exposures to stress are also central to encoding a largely undefined but still well-recognized "resiliency" in medical, military, and law enforcement personnel and other individuals needing to perform well in adverse environments.

## **EXTENDING THE DURATION OF ADAPTIVE CHANGE**

With respect to stimulus frequency influencing the duration of the resultant phenotype, examples can be found in the conditioning literature that either strongly hint at such a possibility or support it directly. For example, the therapeutic window for myocardial ischemic tolerance resulting from a single hypoxic challenge was extended significantly if intermittent mild hypoxia is used as the conditioning stimulus (23, 24). Similarly, long-term thalamic atrophy following transient focal stroke in mice was attenuated by an intermittent hypoxic postconditioning stimulus initiated 5 days after ictus (25). In neonatal (26) and adult rats (27), multi-day hypoxic postconditioning reduced cerebral injury when the first challenge was initiated an hour after the severe hypoxic-ischemic insult. We found that, relative to the <3-days duration of ischemic tolerance induced in the mouse neural retina by a single hypoxic preconditioning stimulus, tolerance lasting at least 4 weeks after the last hypoxic preconditioning treatment can be realized if the hypoxic preconditioning challenge is repeated six times over a 2 week period (28). A similar extension of the therapeutic window (>2 months) for protection against murine cerebral ischemia is also afforded by a 2-week intermittent hypoxia preconditioning regimen (29), but only with stochastic increases in the frequency, duration, and intensity of the hypoxic stimulus relative to that which was efficacious in protecting against retinal ischemia. This distinction, and the examples above, not only suggest that tissue-, cell, age-, and species-dependent hormetic dose–response relationships may be operative but also suggest that stimulus frequency

is not a simple concept in and of itself, and variations in the magnitude and duration of a given intermittently presented stimulus, as well as its temporal pattern, may make the difference between efficacy, impotence, and harm. The mixed preclinical track-record reported to date, wherein some repetitive conditioning protocols promote stroke tolerance, but not necessarily long-lasting stroke tolerance (30, 31), may be a reflection of this complexity. While the concept that extended periods of tolerance resulting from repetitive conditioning can be modeled in neuronal (32) and cerebral endothelial cell (33) cultures, the ultimate identification of a safe and efficacious, intermittent stimulus-based conditioning protocol – either physiologic or pharmacologic – that provides consistent, long-lasting protection in patients will prove extremely challenging in a diverse cross-section of human patients without reliable biomarker metrics to guide dosing regimens. Clearly, at this juncture, continued work in animal models is warranted.

Despite the above caveats, because most heart attacks and strokes are unpredictable, the inherent clinical advantage of postconditioning relative to preconditioning still generates considerable translational excitement. Progress might be accelerated if postconditioning protocols move beyond cyclical interruptions of early reperfusion to assessments of the benefits accruing from more protracted, repetitive treatments, and if long-term survival rates and functional metrics of post-stroke recovery become the more standard endpoints. Indeed, in cardiac ischemia, repeated remote postconditioning improved long-term survival relative to both a single preconditioning challenge and to an even lower frequency of repetitive remote postconditioning (34). Intentionally delaying the start of the repetitive postconditioning treatment volley may be prudent, given that, when initiated a week after stroke, moderate intermittent hypoxia rescued ischemia-induced impairments in learning and memory (35), whereas initiating the same treatment 1–2 days post-stroke increased mortality (36). Similarly, the aforementioned study of intermittent hypoxic postconditioning preventing post-ischemic thalamic atrophy was efficacious when the stimulus volley was initiated 5 days, but not 1 day, after ictus (25). Predictably, the optimal treatment window will likely vary depending on the age, species, injury type, and the postconditioning stimulus employed [including physical rehabilitation and exercise (37, 38)]. No matter what the tissue, rigorous, systematic investigations of multiple postconditioning "dose" regimens/protocols (34, 36, 39) – essentially time-consuming, iterative, titration-directed experiments – will be needed to identify both the dose and the optimal time after the acute injury to initiate treatment if this therapeutic approach is ever to be clinically implemented (40, 41). At present, we really know very little about the frequency of a given postconditioning stimulus needed to activate innate plasticity mechanisms capable of promoting and sustaining a neurorecovery-enhancing phenotype. We know less still about "preconditioning" treatments that effectively "post-condition" the stroked brain against a second stroke; repetitive stimuli may hold promise as the ideal treatment approach for this challenge. That said, some clinical trials of repetitive conditioning have moved forward empirically, and some have shown efficacy. As examples, hyperbaric oxygen treatments (5 days/week for 8 weeks) led to significant neurological improvements in both stroke (42) and head trauma (43) patients, even when the treatment was initiated many

months after injury. And intermittent remote postconditioning by upper limb cuff inflation twice a day for 300 consecutive days proved effective in reducing the incidence of recurrent stroke in patients with symptomatic atherosclerotic intracranial arterial stenosis (44).

#### **CONDITIONING FOR CHRONIC NEUROLOGIC DISEASE**

That distinct patterns of "continuous" but intermittent conditioning treatments provide protracted periods of protection against stroke and other acute neurological disorders implies that the slow and progressive neuronal death that defines neurodegenerative disease might also be significantly impacted by a similar therapeutic strategy. Evidence from the hormesis field that repeated presentations of low-dose stressors extend beneficial, prevention-, and/or recovery-promoting effects in preclinical models of more chronic cardiovascular, immunological, metastatic, and other non-neurological and non-psychological pathologies, as well as in human studies (4), supports this contention. Not surprisingly then, when tested, the intermittent hypoxia protocol that we leveraged in mice to extend the duration of tolerance to acute retinal ischemia (28) also proved efficacious as a preconditioning stimulus against glaucoma, significantly reducing retinal ganglion cell somal and axonal loss at 3 and 10 weeks of disease in mice (45). Moreover, significant improvements in these same cell survival metrics were realized when we postconditioned mice with a repetitive intermittent hypoxia protocol initiated after disease onset (46). Repetitive postconditioning with brief bouts of retinal ischemia was also efficacious in reducing retinal neurodegeneration in rat models of glaucoma (47) and diabetic retinopathy (48). Suggestive of potential efficacy in Alzheimer's, intermittent hypoxic postconditioning reduced oxidative and nitrosative stress metrics and improved morphologic and functional (memory) outcomes following intracerebral injections of amyloid beta (49). Although not widely realized, an extensive number of preclinical and clinical studies in the Soviet Union, in which an estimated two million individuals were treated over long periods of time with intermittent hypoxia, document a variety of health-promoting and injury-resistant outcomes from such treatments, in the absence of adverse effects (50). Collectively, these findings provide strong support for the hypothesis that intermittent conditioning during the progression of chronic neurological disease may provide a means of both inducing and "holding on" to an adaptive phenotype that in turn is manifested not only as a reduction in the kinetics of disease progression but also as improvements in functional status and long-term survival.

#### **LIFESPAN EFFECTS**

As alluded to above, the idea that repetitive stress conditioning can trigger and possibly maintain long-lasting phenotypic change in the CNS actually derives strong historical support from the neuropsychology and behavior fields, which provide rich examples of sustained plasticity within and across age groups, and ideal opportunities for considering stress along a negative–positive continuum, with distress anchoring one end and "eustress" anchoring the other (51). For instance, when initiated during adulthood, repeated mild stress exposures lead to persistent decreases in behavioral disorders and improvements in memory (52, 53). And

the literature is actually replete with studies documenting adaptive phenotypes persisting into adulthood as a result of intermittent, mild stress challenges only experienced by the individual during intra-uterine or post-uterine development. This concept was introduced over 60 years ago based on the serendipitous findings of Levine (54), and is somewhat akin to the immunological concept of early childhood vaccinations for conferring life-long protection against disease. As it turns out, most research over the ensuing decades has focused on documenting illness phenotypes secondary to child or adolescent stress in an attempt to understand the origins of psychiatric disease (55). Nevertheless, reflecting yet another manifestation of "the dose makes the poison" and new ways of thinking about evolutionary fitness, there are still some investigations to be found of beneficial adult phenotypes resulting from exposing the same individual, when much younger, to a given stressor and/or exposing their caregiving mothers to stress (56, 57). Hypoxia-related examples of the former include the finding that postnatal mice exposed from birth to 4 weeks of age to mild intermittent hypoxia exhibit improved spatial learning and memory during their adult life (58), and the report that brief hypoxic challenges during neonate life confer resistance to senescence and better preservation of cognitive function in aged rats (59). Resilience to stress in adult mice raised by mothers with access to postpartum exercise (60) is an example of the latter. Intermittent separations of baby monkeys from their mother may represent a form of "stress inoculation" that leads to enhanced arousal regulation and resilience of these offspring in later life (15). There is also evidence that unpredictable, stochastic stress exhibits unique agedependent effects, promoting future resilience when experienced by juvenile animals, but not by adults (61). Given these and other provocative findings, some have even proposed – for humans – intentional exposures to intermittent eustress (like mild hypoxia) during neonatal or adolescent periods as a way to prevent or lessen future disease burden (62).

#### **EPIGENETICS AS PRIMARY MECHANISM**

The molecular basis of the many adaptive responses to repeated stress highlighted thus far is predictably complex, given the relative permanence of the resultant phenotype. Many studies have sought to characterize the beneficial phenotypes, finding evidence for increased neurogenesis (53), changes in hormonal balance (15, 18), changes in modulators of synaptic plasticity (63, 64), changes in sodium–calcium exchangers (NCX) (65), and elevated levels of HIF gene target mRNAs/proteins (52) or other survival factors (31), to name a few. While identifying these phenotypes is interesting with respect to understanding how disease resistance is ultimately manifested, it is epigenetics that deserves attention as the fundamental mechanism responsible for the long-lasting responses to repetitive conditioning stimuli, and thus the likely target of future therapeutics. In brief, epigenetics involves the regulatory processes – DNA methylation and changes in chromatin structure secondary to post-translational histone modifications – that reside "above" the level of genes and control their readout. During development, epigenetics specifies cell fate determination and perpetuation, but we now know that these same mechanisms are engaged throughout the lifespan by "experience" or "environment," in all their different forms. Importantly, considerable evidence indicates that the changes in gene expression resulting from these stable covalent DNA modifications or epigenetic "marks" can be long-lasting, and, in some instances, endure throughout the lifetime of post-mitotic cells; some may even persist through cell division and be transmitted via the germline to future generations (see below). Thus, epigenetics is really the biochemically driven interface between nature (genes) and nurture (all manner of environmental and behavioral/psychological stresses or exposures). Proximal to this interface, so to speak, distinct features of an experience or a stressor (e.g., frequency, severity, etc.) that, as alluded to earlier we have only recently begun to dissect and define with respect to threshold and interactive effects, dynamically modify the epigenome. Moreover, epigenetic marks can accumulate over the course of multiple exposures and then act collectively to determine a new homeostatic phenotypic set-point. Distally, after encoding molecular memories of these experiences and exposures, CNS function and behavior is altered secondary to changes in gene expression, thus impacting one's vulnerability or resilience to future stressors/disease (66). In essence, epigenetics is really the "hard-wired" evolutionary response for successful adaptation to changing natural and social environments. With the realization that phenotype is fluid, and not rigidly defined by genotype, it is not an exaggeration to claim that broadening our understanding of "neuroepigenetics" (67) and its regulation at both cellular and organismal levels holds incredible promise for reducing CNS disease burden and enhancing neurological health.

The ischemia-tolerant phenotype resulting from a pre- or postconditioning stimulus is, by definition, an epigenetic response, but only recently have we begun to directly recognize it as such (68–70). Genomic and proteomic analyses consistently reveal that the ischemia-tolerant CNS is defined by a broad transcriptional repression (6, 68, 71) – in effect, the manifestation of an epigenetically mediated response to the conditioning stimulus. Tests of the hypothesis that members of the evolutionarily conserved polycomb protein family – known repressors of gene transcription secondary to their ability to posttranslationally modify histones and thus maintain them in an inactive state (72) – may be causal epigenetic mediators ultimately responsible for establishing this metabolically downregulated phenotype confirmed that increases in polycomb group proteins define the "traditional" ischemia-tolerant CNS (68, 73). Certainly, other transcriptional regulators of gene repression – such as repressor element-1 silencing transcription factor (REST) (74) – may also be involved in orchestrating this response. Whether polycomb and/or REST proteins and/or others participate in mediating the longer periods of injury resistance induced by repetitive pre- or post-conditioning stress paradigms has yet to be examined. Additional studies uncovering other epigenetic features of conditioning-induced tolerance have been forthcoming (75).

A rapidly evolving subfield of epigenetics extends the aforementioned phenomenon of even life-long phenotypic change within an individual to the transmission of adaptive or maladaptive phenotypes from parents to their offspring. Epigenetic inheritance, or transgenerational epigenetics, provides a mechanistic foundation for Lamarck's notion that the effects of environment and/or experience could be inherited by, and thus benefit, one's immediate offspring – known as "soft inheritance" in Lamarck's day. This

phenomenon is not inconsistent with Darwinian evolutionary theory, given the selection pressure inherent in preserving adaptive phenotypic variation if it increases fitness when resources and/or other environmental stress levels change; but it does carry the important implication that random DNA mutations are not the sole driving force of evolution. In transgenerational epigenetics, gene expression is altered, not genetic inheritance; environment becomes heredity. At least in theory, this suggests the possibility of obtaining desired phenotypes in offspring that prevent or reduce disease burden and/or enhance vitality by therapeutically manipulating epigenetic regulatory systems in their parents. Conditioning across generations might seem at first blush to represent an implausible extension of tolerance's therapeutic window. However, while the vast majority of preclinical studies have focused on the germline linking of adverse or dysfunctional behavioral changes in an individual to life experiences of their parents or grandparents (76–78), a small number of reports to date provide convincing examples, in mammals, of the transgenerational persistence of beneficial, epigenetically acquired phenotypes (79–82). Moreover, there is very intriguing epidemiological evidence that this occurs in humans (80, 83–85). Obviously, understanding the mechanistic basis of both potential outcomes portends huge repercussions for evolutionary biology, and will likely change the face of our understanding of the genetic basis of neurological disease.

#### **CONCLUSION**

Despite the discovery of the robustness of endogenous cytoprotection almost 25 years ago, the thousands of published preclinical successes, and even the dozens of efficacious studies in humans, the time-limited therapeutic window that has inadvertently come to define conditioning-based responses continues to constrain its acceptance as a viable therapeutic strategy, even for acute tissue injury. However, given the accumulating evidence supporting long-lasting, epigenetics-mediated changes in phenotype in the CNS and other tissues secondary to repetitive stress, perhaps it is time to shelf the assumption that conditioning-induced tolerance is "transient," reevaluate our assumptions about the defining features of tolerance, and in so doing open new doors regarding its full clinical destiny. As scientists and physicians, capturing the ability to leverage sustained, beneficial phenotypic changes capable of providing protracted periods of resilience to acute and chronic neurological injury should be our next collective goal.

#### **ACKNOWLEDGMENTS**

Studies by the author mentioned in this review were supported by: NIH EY18607, AHAF/BrightFocus National Glaucoma Foundation, EY02687 (Department of Ophthalmology, Washington University), NIH Neuroscience Blueprint Interdisciplinary Core Grant P30 NS057105 (Washington University), the HOPE Center for Neurological Disorders (Washington University), and the Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International.

#### **REFERENCES**

and preconditioning stress within a hormetic dose-response framework. *Toxicol Appl Pharmacol* (2007) **222**(1):122–8. doi:10.1016/j.taap.2007.02.015

and postconditioning. *Adv Exp Med Biol* (2013) **961**:223–40. doi:10.1007/978- 1-4614-4756-6\_19

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 06 January 2015; accepted: 18 February 2015; published online: 02 March 2015.*

*Citation: Gidday JM (2015) Extending injury- and disease-resistant CNS phenotypes by repetitive epigenetic conditioning. Front. Neurol. 6:42. doi: 10.3389/fneur.2015.00042*

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology.*

*Copyright © 2015 Gidday. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Histone deacetylases exert class-specific roles in conditioning the brain and heart against acute ischemic injury**

## *Sverre E. Aune, Daniel J. Herr, Craig J. Kutz and Donald R. Menick\**

*Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC, USA*

Ischemia-reperfusion (IR) injury comprises a significant portion of morbidity and mortality from heart and brain diseases worldwide. This enduring clinical problem has inspired myriad reports in the scientific literature of experimental interventions seeking to elucidate the pathology of IR injury. Elective cardiac surgery presents perhaps the most viable scenario for protecting the heart and brain from IR injury due to the opportunity to condition the organs prior to insult. The physiological parameters for the preconditioning of vital organs prior to insult through mechanical and pharmacological maneuvers have been heavily examined. These investigations have revealed new insights into how preconditioning alters cellular responses to IR injury. However, the promise of preconditioning remains unfulfilled at the clinical level, and research seeking to implicate cell signals essential to this protection continues. Recent discoveries in molecular biology have revealed that gene expression can be controlled through posttranslational modifications, without altering the chemical structure of the genetic code. In this scenario, gene expression is repressed by enzymes that cause chromatin compaction through catalytic removal of acetyl moieties from lysine residues on histones. These enzymes, called histone deacetylases (HDACs), can be inhibited pharmacologically, leading to the de-repression of protective genes. The discovery that HDACs can also alter the function of non-histone proteins through posttranslational deacetylation has expanded the potential impact of HDAC inhibitors for the treatment of human disease. HDAC inhibitors have been applied in a very small number of experimental models of IR. However, the scientific literature contains an increasing number of reports demonstrating that HDACs converge on preconditioning signals in the cell. This review will describe the influence of HDACs on major preconditioning signaling pathways in the heart and brain.

**Keywords: ischemia-reperfusion injury, histone deacetylase inhibitors, stroke, posttranslational modification, enzymatic crosstalk, preconditioning, postconditioning, reperfusion injury salvage kinase**

# **Introduction**

Worldwide, 33 million people suffer a stroke each year (1). Ischemia, which can occur in all tissues, is defined as the stress that a tissue experiences when both oxygen and substrate are reduced (2). Stroke, defined as insufficient blood flow to the brain, is one major type of cerebral ischemia and may cause transient to permanent loss of brain function or death (3).

#### *Edited by:*

*Giuseppe Pignataro, University of Naples Federico II, Italy*

#### *Reviewed by:*

*Akihiko Urayama, University of Texas Houston Medical School, USA Maria Jose Sisalli, University of Naples Federico II, Italy*

#### *\*Correspondence:*

*Donald R. Menick, Gazes Cardiac Research Institute, Medical University of South Carolina, 114 Doughty Street, Room 331, Charleston, SC 29425, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology*

> *Received: 14 February 2015 Accepted: 15 June 2015 Published: 30 June 2015*

#### *Citation:*

*Aune SE, Herr DJ, Kutz CJ and Menick DR (2015) Histone deacetylases exert class-specific roles in conditioning the brain and heart against acute ischemic injury. Front. Neurol. 6:145. doi: 10.3389/fneur.2015.00145*

In a subset of patients (*<*15%), stroke occurs as a result of cardiac arrest and cardiac surgery; secondary stroke is, by definition, the result of embolic events originating in the heart (4). In spite of intense research effort directed toward reducing the impact of these conditions, world rates of morbidity and mortality as a result of brain and heart disease continue to rise, most strikingly in developing countries (5). The lack of broadly effective treatment continues to fuel the search for new molecular targets in ischemia-reperfusion injury (IRI). Intriguingly, studies of molecular pathology in the brain and heart are often informed by cancer research. Recent clinical advances have revealed the efficacy of using small molecule inhibitors of histone deacetylases (HDACs) to target malignancy (6). HDACs are enzymes which control signal transduction and gene expression in all cell types (7). Here we review the experimental evidence in support of applying HDAC inhibitors in settings of cerebral and cardiac ischemia, with emphasis on the roles that HDACs play in signaling events that occur as a result of IRI. Importantly, this nascent experimental work indicates that HDAC inhibitors show great promise for treating patients at high risk for stroke or cardiac arrest and for patients electing to receive brain and heart surgeries.

Endogenous tolerance to ischemia can be evoked in the heart and brain (4, 8). Classical ischemic conditioning in a tissue requires the mechanical application of several momentary, sublethal reductions in oxygen and substrate delivery, which reduce the injury caused by a more severe ischemic insult (9). Organs subjected to classical ischemic conditioning experience only transient stimulation of endogenous protective mechanisms, which limits application of the conditioning stimulus to shortly before prolonged insult, as in preconditioning a patient of elective surgery, or to shortly afterward, as in postconditioning a patient of out-ofhospital stroke or cardiac arrest. The idea that similar magnitudes of endogenous protection can be evoked pharmacologically is a considerable advancement over the barrier of time-dependence instituted by mechanical ischemic conditioning (10). It follows that drug-evoked conditioning is a promising treatment strategy for patients undergoing elective brain or heart surgery, or those at high risk of stroke or cardiac arrest (11).

Prevention of cell death is the primary effect of ischemic conditioning (12, 13). Many forms of ischemic conditioning (including classical, pharmacological, and remote conditioning) converge on signaling pathways that either inhibit cell death or activate endogenous cell survival maintenance programs. Once active, cell survival programs improve endoplasmic reticulum stabilization (14), increase expression of antioxidant enzymes (SOD2; catalase) (15), inhibit endogenous cell death programs (Bax/Bad/Bcl2; c-jun) (16), activate transcription of genes for repair enzymes (Hsp70; HIF-1-alpha) (17, 18), and stimulate autophagic flux (HDAC6) (19).

Acute adaptation to ischemia through conditioning stimuli is mediated by protein posttranslational modifications (PTMs) (20, 21). Historically, protein phosphorylation has been the most rigorously characterized of the PTMs in settings of experimental ischemic conditioning. These include the reperfusion injury salvage kinases (RISK) pathways of Raf/MEK/ERK1/2 and PI3K/Akt/eNOS, the JAK/STAT transcriptional pathways and the calcium-responsive PKC pathways (22–25). However, accumulating evidence shows that protein acetylation also plays major roles in regulating cell survival through ischemic conditioning (7).

# **Histone Deacetylases Regulate Cell Fate in Cerebral Ischemia**

Histone deacetylases are a class of epigenetic enzymes that have come under recent intense scrutiny as pharmacological targets for patients suffering stroke. HDACs remove acetyl moieties from *ε*-amino groups of lysine residues on histones and non-histone proteins (25). Deacetylation of histones enhances chromatin compaction, which renders DNA less available for binding by regulatory factors leading to repression of gene expression (26, 27). In this function, HDACs exert classical transcriptional control. This process is reversible via enzymatic histone acetyltransferase (HAT) activity. The zinc-dependent HDACs have been divided into classes based on homology to yeast transcriptional repressors. Class I comprises HDACs 1, 2, 3, and 8; class IIa comprises 4, 5, 7, and 9; class IIb comprises 6 and 10; and class IV comprises HDAC11 (7). Class III, called sirtiuns, are NAD+-dependent and will not be discussed in this review, but have been expertly reviewed elsewhere (28). HDAC enzymes are widely expressed in rodent brains, and are localized to specific cellular compartments in isoform-specific patterns (29, 30). Class I HDACs are generally restricted to the nucleus where they impose transcriptional control, whereas class IIa HDACs transit the nuclear membranes and enter the cytoplasm in a process mediated by phosphorylation (31). Furthermore, HDAC6 is primarily, though not constitutively, cytoplasmic (19).

As mentioned above, HDACs deacetylate both histone and non-histone proteins. When HDACs deacetylate histones and repress gene transcription, cell survival is impacted after several hours and days on time scales necessary for protein expression (7). Non-histone deacetylation is another element of this complex code of enzymatic crosstalk, which is distinct from direct inhibition of gene expression by histone deacetylation. Importantly, the acetylation state of a given metabolic signaling factor may mediate its phosphorylation, methylation, and ubiquitination state, thereby determining its subcellular location, activation, or degradation, with immediate implications for cell survival in the seconds to minutes following the insult (32). The acetylation state of transcription factors, co-activators, and co-repressors can regulate their activity (33). Decoding the complex patterns of HDAC enzymatic crosstalk will enhance our understanding of histone and non-histone protein lysine deacetylation and its impact on the survival of cells under ischemic stress (34).

Importantly, experimental cerebral ischemia causes upregulation of class I/IIb HDAC expression, which possibly implicates them in ischemic pathology (29). For example, HDAC1 must exist in complex with HDAC3 to promote apoptosis in cerebellar granule neurons; the toxic effects of HDAC1:HDAC3 association were mitigated by activation of PI3K/Akt signaling (35). Cortical neurons transfected with HDAC3 or HDAC6 shRNA each exhibited decreased apoptosis when exposed to prolonged oxygen–glucose deprivation (30). Furthermore, HDAC2 mutant mice exhibited reduced retinal degeneration following ischemia Aune et al. Histone deacetylases in ischemic conditioning

(36). In response to these findings and many others, a variety of pharmacological HDAC inhibitors have been developed and applied in animal models of cerebral ischemia. Results from these studies have revealed that HDAC inhibitors can pharmacologically condition the neuron against ischemic injury through de-repression of transcription (37, 38). While these studies suggest that pharmacological inhibition of class I/IIb HDAC isoforms (so-called "pan-inhibitors") promote survival of stressed neurons, the evidence reviewed below indicates that the catalytically inactive IIa isoforms may be beneficial to cell survival from IRI.

# **Small Molecule HDAC Inhibitors Condition the Brain Against Ischemic Injury**

Reduction in infarct volume and prevention of apoptosis are the most obvious physiological effects of class I/IIb HDAC inhibitors in experimental cerebral ischemia (see **Table 1**). The molecular mediators are numerous but consensus is forming around certain cellular processes: modulation of apoptotic intermediates (caspases, Bcl-2), stabilization of the cellular stress response (Hsp-70, EIF-2α, CHOP), transcription of oxygen-responsive enzymes (HIF-1α, Nrf2), regulation of calcium handling (BDNF, CREB), and activation of survival kinase cascades (Akt, ERK, AMPK, p21).

Administration of the mood stabilizer and weak HDAC inhibitor valproic acid (VPA) after permanent right carotid artery occlusion prevented neuronal apoptosis in a dose-dependent manner in neonatal rats (39). Rats treated with VPA during middle cerebral artery occlusion (MCAo) exhibited reduced infarct volume, enhanced angiogenesis (40), neurogenesis (41, 42), and reduction of monocyte infiltration (43). These effects were correlated with increased transcription of Hsp70 (43, 44), HIF1 alpha and MMP-2/9 (40), or GLT-1, a transporter protein, which accelerates clearance of glutamate in damaged gray and white matter (41). VPA protected retinal ganglion cells from ischemiareperfusion (IR) through reduction of mitochondria-mediated apoptosis (45) and endoplasmic reticulum stress-induced apoptosis (46), and enhanced Hsp70 promoter acetylation in cortical neurons through inhibition of a class I HDAC (47). The potent hydroxamate trichostatin A (TSA) decreased infarct volume in rodents given MCAo, which depended on induction of Hsp70, Bcl-2, and Akt phosphorylation, or on gelsolin, an essential regulator of actin homeostasis (43, 48, 49). Mice treated with TSA at the onset of permanent MCAo exhibited reduced infarct volume through increased Nrf2-dependent transcription of antioxidant enzymes (50). TSA also reduced transcription of inflammatory proteins MMP-1 and MMP-3, and reduced caspase-3 activation up to 24 h after the onset of ischemia in the retina (51, 52). Sodium butyrate (a potent analog of VPA) treatment before permanent MCAo evoked a 50% reduction in infarct volume through increased expression of p21, a cyclin-dependent kinase inhibitor, which prevents pro-apoptotic gene transcription (53–55). Furthermore, mice treated with 4-phenylbutyrate before or after ischemia–hypoxia exhibited reduced ER-stress-mediated apoptosis through reduction of EIF2-alpha phosphorylation (56). Mice treated with the potent hydroxamate Vorinostat (FDA approved for treatment of T-cell lymphoma) at the onset of MCAo exhibited reduced infarct size and increased transcription of Hsp70 and Bcl2 (57, 58). Furthermore, neurons exposed to oxygen–glucose deprivation *in vitro* and mice subjected to MCAo *in vivo* exhibited increased acetylation at the Bcl-xL promoter when treated with Entinostat, a class I selective HDAC inhibitor; the effect was mediated by enhanced NF-kB p50 acetylation and decreased activation of the Bim promoter (59).

While class I HDACs seem to play pathological roles in cerebral ischemia, there is evidence that class IIa HDACs are required for cell survival following neuronal stress. Genetic heterogeneity surrounding the *HDAC9* gene is associated with large vessel ischemic stroke (60). By directly inhibiting the c-jun promoter, HDAC4 (61) and HDAC7 prevented neuronal cell death induced by low potassium (62). HDAC4 is required for the normal development of retinal neurons through the stabilization of HIF-1-alpha (63). HDAC4 and HDAC5 knock-in protected neuron-like pheochromocytoma cells from apoptosis induced by OGD, which was partly dependent on HMGB1 activity (64). Conversely, nuclear export of HDAC5 was required for regeneration after acute axonal injury, a condition that promotes rapid influx of calcium (65). In fact, nuclear calcium levels regulate the association of class IIa HDACs with a MEF2-SMRT corepressor complex (66– 68). Given this, it is possible that class IIa HDACs may correct calcium-induced pathological gene expression in neuronal ischemia.

# **HDAC Enzymatic Crosstalk in Cerebral Ischemia**

Evidence is accumulating that HDAC signal transduction pathways communicate in crosstalk with kinase signal cascades in cerebral ischemia. The ability of HDAC inhibitors to condition the neuron in the seconds to minutes following acute ischemic stress may be dependent on the concurrent activity of certain cell survival kinases. As mentioned above, TSA prevented oxidative cell death in cortical neurons through increased transcription of p21, which inactivates pro-apoptotic c-jun transcription by inhibiting the kinase ASK-1 (53–55). HDAC3 was phosphorylated by GSK3 beta and was required for cell death induced by low potassium in cultured cortical neurons; neuronal death was prevented by pharmacological inhibition of GSK3-beta, and with constitutively active Akt, a known inhibitor of GSK3-beta (69). Conversely, the class IIa HDAC4 protects neurons from cell death induced by low potassium by direct inhibition of cyclin-dependent kinase-1 activity, independent of PI3K/AKT, c-jun, or RAF/MEK/ERK signaling (61).

PI3K and AKT activities are both required for the neuronal conditioning achieved with VPA (47). Interestingly, induction of Hsp70 by VPA and other Class I HDAC inhibitors resulted in increased histone methylation in primary neurons and astrocytes (70). In particular, as confirmed by chromatin immunoprecipitation, HDAC inhibition caused increased methylation at the Hsp70 promoter, a histone landscape favoring transcriptional activation. This suggests an intricate interplay between histone acetylation

#### **TABLE 1 | Physiological effects of HDAC inhibitors in experimental models of stroke**.

*RCAo, right carotid artery occlusion; pMCAo, permanent carotid artery occlusion; ONC, optical nerve crush; Ac, acetylated; OGD, oxygen–glucose deprivation.*