*3.9.2. CH stability*

The time during which a node is in the state of a CH determines stability. It is the mean time duration of the nodes, remaining its leadership role as CHs. Long CH lifetime implies, few changes and good stability. The information is disseminated by groups enhancing the communication delay, reliability, low data delivery and congestion issues, making the vehicular networks accurate and efficient. CH stability can be affected by different factors such as merging, distance between CHs, exit from the road etc. In VANETs, merging collisions can happen among vehicles moving in the same direction due to acceleration or deceleration, it is more likely to occur among vehicles moving in opposite directions (approaching each other) or between a vehicle and a stationary RSU since they approach each other with a much higher relative velocity as compared to vehicles moving in the same direction. The high mobility of the shifting nodes within the networks causes lots of challenges to face and affects stability.

If vehicles are changing their state very often in intersection scenarios and stay only for short times in the CH state, CH stability is low. In some of intersection based approaches the first vehicle to enter the intersection region in a particular direction is elected as CH to improve stability. Furthermore, some cluster-based routing algorithms, the selection of CH are based on willingness factor which defines the relative stability of a node. CH stability is also based on the threshold distance between the two CHs. Optimal distance between two CHs should be obtained.


*2.8. Statistical Analysis*

All experiments were conducted with at least three independent replicates. The results were analyzed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was determined by student's *t*-test if two groups were compared. A *P* value less than 0.05 was considered statistically significant.


**Acknowledgements**

The authors are grateful for the financial support for this research to the Secretariat of Science and Technology of the National University of La Rioja (Project N ° 170/2016 "Stratigraphic Study of the Group Alto de San Nicolás del Miocene, Campo de Talampaya, The Rioja") and the authorities of the Parque Nacional Talampaya.


**Localization, Mapping and SLAM in Marine and Underwater Environments**

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## Corticosteroids A Paradigmatic Drug Class

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(IGF-1) whose levels decrease with age. IGF-1 infusion following stroke, which prevents estrogen neurotoxicity in middle-aged female rats, altered the expression of two conserved IGF pathway regulatory miRNAs, Let-7f, and miR-1. Adult female rats treated with anti-miR-1 as late as 4 h after ischemia had significantly reduced cortical infarct volume, and anti-Let-7 robustly reduced both cortical and striatal infarcts and preserved sensorimotor function and inter-hemispheric neural integration (117). However, anti-Let-7 was only effective in intact females and had no effect on males or ovariectomized females, indicating that the gonadal steroid environment critically modifies miRNA action (117). Further studies examined the effect of post-stroke IGF-1 treatment on miRNA expression in middle-aged female rats (118). IGF-1 administered immediately after ischemia significantly reduced infarct volume in 9- to 11-month female rats 24 h after stroke. Post-stroke IGF-1 treatment significantly downregulated 8 out of 168 disease-related miRNAs in tissue isolated 4 h after ischemia. KEGG pathway analysis predicted that targets of these miRNAs include proteins involved in PI3K–Akt signaling, cell adhesion/ECM receptor pathways, and T-and B-cell signaling. Further, Phospho-Akt was reduced by 4 h after ischemia, but was elevated by IGF-1 treatment at 24 h after ischemia. IGF-1 also induced Akt activation, an effect that was preceded by a reduction of BBB permeability and global suppression of cytokines at 4 h post-stroke (118).

## **CONCLUSION**

The goal of this review was to present the current state of knowledge regarding ncRNAs and their role(s) in stroke and neuroprotection. However, of these three relatively new families of ncRNAs, miRNAs are the most well studied to date. Yet the lncRNAs and piRNAs are certain to contribute to the setting of ischemia and neuroprotection, and there are several important future lines of investigation for all ncRNA studies. The foregoing studies highlight the difficulty of analyzing miRNA expression data, due to the diversity of ischemia and neuroprotection models, the time points examined, and the fact that since miRNAs can act in concert to exert effects on target mRNAs, focus on a single miRNA is unlikely to realistically capture endogenous responses. Thus, the first such studies must focus on ways to meaningfully translate the outcome of multiple expression profiling studies from diverse labs in order to identify global signaling pathways or targets that have been underappreciated to date. To address this issue, Xiao and colleagues sought to model complex interactions in order to identify synergistic effects within miRNA responses to brain ischemia (119). miRNA array expression data submitted to the Gene Expression Omnibus database from multiple studies in different labs were examined to identify mouse brain miRNAs differentially expressed between ischemia and control. Common predicted targets of the miRNAs were then used as a candidate subset to identify functional modules. An miRNA functional synergistic network, constructed by assembling all miRNA synergistic pairs, revealed 51 differentially expressed miRNAs identified between ischemia and control (32 upregulated and 19 downregulated). Among them, 24 miRNAs commonly regulating at least one target gene were used to construct a network. Subsequent analyses revealed 16 miRNAs forming 20 miRNA

Saugstad Non-coding RNAs in stroke and neuroprotection

interaction pairs that participated in inflammatory responses, suggesting these 16 miRNAs as underlying targets for prevention and/or treatment of stroke (119). These analyses highlight that the power of global miRNA and ncRNA studies lies in their potential to identify proteins or pathways involved in responses to ischemia. The second such studies must address the need to identify the effects of ncRNAs acting in concert to regulate cellular phenotypes or responses to stress. To date, each family of ncRNA has largely been examined individually, due to technical barriers to combinatorial analysis of all ncRNAs. RNAseq studies can be used to identify all RNA species in a given sample set, but even this method is limited in the ability to analyze distinct sizes of RNA species. Thus, lncRNAs (>200 nt) are not yet typically sequenced together with small ncRNAs such as the miRNAs and piRNAs (~20–36 nt). A third line of exciting studies will come from the not-so-recent finding that extracellular RNAs exist in biofluids, mostly contained within EVs such as microvesicles, and can serve as biomarkers of neurological disorders (120). EVs and their molecular components are increasingly shown to be effectors of cell–cell communication in the brain and their uptake can alter the phenotype of a recipient cell (32). Thus, future lines of investigation should include examining the consequences of disrupted blood flow and metabolism, such as during a stroke, on communication mediated by EVs. Further, given that EVs mediate communication between distinct cell types in the brain, future therapeutic strategies for the treatment or prevention of stroke and other brain disorders might include focused delivery of molecular or pharmaceutical agents by EVs, potentially using vesicles isolated from ones own biofluids. The importance and excitement of such studies is highlighted by the recent formation of the Extracellular RNA Communication program (http://commonfund.nih.gov/Exrna/index) supported by the National Institutes of Health Common Fund that aims to discover fundamental biological principles about the mechanisms of exRNA generation, secretion, and transport; to identify and develop a catalog of exRNA in normal human body fluids; and to investigate the potential for using exRNAs as therapeutic molecules or biomarkers of disease. Thus, there are many new and exciting future opportunities for research and development in the field of ncRNAs, and in particular for their role in, contribution to, and regulation of the treatment of human diseases, including stroke.

#### **ACKNOWLEDGMENTS**

I sincerely thank Dr. Michael Roberts and Dr. Theresa Lusardi for their excellent comments and editorial contributions to this manuscript. While all efforts were made to provide a comprehensive review on this topic, the author sincerely apologizes for any studies that may have been inadvertently overlooked during creation of the manuscript. Supported by National Institutes of Health R01NS064270 and R21NS082832.

#### **REFERENCES**

1. Vemuganti R. All's well that transcribes well: non-coding RNAs and poststroke brain damage. *Neurochem Int* (2013) **63**:438–49. doi:10.1016/j.neuint. 2013.07.014

by sevoflurane preconditioning. *CNS Neurol Disord Drug Targets* (2013) **12**:381–91. doi:10.2174/1871527311312030011

role in the development of permanent focal cerebral ischemia. *Stroke* (2004) **35**:2566–70. doi:10.1161/01.STR.0000143730.29964.93

for middle-aged female rats. *PLoS One* (2014) **9**:e91427. doi:10.1371/journal. pone.0091427

**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: 30 January 2015; paper pending published: 10 February 2015; accepted: 23 February 2015; published online: 13 March 2015.*

*Citation: Saugstad JA (2015) Non-coding RNAs in stroke and neuroprotection. Front. Neurol. 6:50. doi: 10.3389/fneur.2015.00050*

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

*Copyright © 2015 Saugstad. 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.*

# Role of MicroRNAs in innate neuroprotection mechanisms due to preconditioning of the brain

### Eva M. Jimenez-Mateos \*

Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland

Insults to the brain that are sub-threshold for damage activate endogenous protective pathways, which can temporarily protect the brain against a subsequent harmful episode. This mechanism has been named as tolerance and its protective effects have been shown in experimental models of ischemia and epilepsy. The preconditioning-stimulus can be a short period of ischemia or mild seizures induced by low doses of convulsant drugs. Gene-array profiling has shown that both ischemic and epileptic tolerance feature large-scale gene down-regulation but the mechanism are unknown. MicroRNAs are a class of small non-coding RNAs of ∼20–22 nucleotides length which regulate gene expression at a post-transcriptional level via mRNA degradation or inhibition of protein translation. MicroRNAs have been shown to be regulated after non-harmful and harmful stimuli in the brain and to contribute to neuroprotective mechanisms. This review focuses on the role of microRNAs in the development of tolerance following ischemic or epileptic preconditioning.

#### Edited by:

Giuseppe Pignataro, Federico II University of Naples, Italy

#### Reviewed by:

Varun Kesherwani, University of Nebraska Medical Center, USA Antonio Vinciguerra, Federico II University of Naples, Italy

#### \*Correspondence:

Eva M. Jimenez-Mateos, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 31 December 2014 Accepted: 23 March 2015 Published: 21 April 2015

#### Citation:

Jimenez-Mateos EM (2015) Role of MicroRNAs in innate neuroprotection mechanisms due to preconditioning of the brain. Front. Neurosci. 9:118. doi: 10.3389/fnins.2015.00118 Keywords: preconditioning, microRNAs, ischemia, epilepsy, neuroprotection

# Preconditioning

The concept of preconditioning was first described in a model of heart ischemia. Here, brief ischemic episodes protected against a subsequent ischemic insult (Murry et al., 1986), opening a new way for the treatment of heart diseases. Several preconditioned treatment paradigms have been used in the clinic (including remote preconditioning stimulus) to protect patients against an ischemic insult in heart pathologies (McCafferty et al., 2014).

 The fact that any injury to the brain applied below the threshold of cell damage, including seizures, will induce preconditioning and neuroprotection to the brain shows the importance of understanding the mechanism underlying preconditioning and its potential as novel treatment option in brain disorders.

The preconditioning stimulus induces a transient state of protection which is called tolerance. Early work demonstrated that de novo protein synthesis was required for tolerance; this implied regulation of gene expression in a time-specific manner and having an effect only several days after the precondition stimulus (Barone et al., 1998).

Preconditioning can induce neuroprotection over two time-frames: (1) Rapid tolerance, which happens in a short period of time and is independent of protein production and is associated with synapse remodeling (Meller et al., 2008). (2) Delayed (classical) tolerance, which evolves over 1–3 days post-preconditioning and requires de novo protein production which peaks after 3 days and diminishes over the course of the following week (Stenzel-Poore et al., 2007).

# Preconditioning Stimuli

Preconditioning can be induced by several stimuli in the brain, including non-injurious ischemia, cortical spreading depression, brief episodes of seizures and low doses of endotoxins (LPS, lipopolysaccharide) (Kitagawa et al., 1990; Simon et al., 1993; Kobayashi et al., 1995; Chen and Simon, 1997; Towfighi et al., 1999). A cross-tolerance phenomenon has been recognized where stimuli and challenge are from different nature. For example, a brief-seizure stimulation or LPS injection can protect against a subsequent ischemic injury (Plamondon et al., 1999; Towfighi et al., 1999).

Data has shown that the reprogramming of genes involved in cellular response to excitotoxic insults plays an important role in the preconditioning process of different tissues (Mirrione et al., 2010). Evidence from genomic studies has demonstrated that diverse stimuli which trigger neuroprotection achieved by preconditioning may share a common process which depends on a fundamental reprogramming of the response to injury, e.g., in ischemia or epileptic tolerance has been shown that 80% of genes are down-regulated. This reprogramming process can induce novel neuroprotective pathways which in turn lead to the synthesis of new proteins changing the molecular and genetic response to subsequent injury (Stenzel-Poore et al., 2004, 2007).

In experimental models of status epilepticus, several preconditioning stimuli including bicuculline, electroshock, kindling, and low dose injection of the convulsant kainic acid, has shown to reduce brain damage after status epilepticus (Amini et al., 2014). Gene profiling has shown that down-regulation of gene expression is a general mechanism underlying the tolerance process (Borges et al., 2007; Jimenez-Mateos et al., 2008; Meller et al., 2008; Della-Morte et al., 2012), including downregulation of genes involved in ion channels, calcium signaling and excitability (Jimenez-Mateos et al., 2008).

# microRNAs

The term MicroRNA, previously called small temporal RNAs (stRNAs) due to their role in the regulation of developmental timing of Caernohabditis elegans, was first introduced in 2001 (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Originally microRNAs were defined as small non-coding RNAs (∼20–22 nucleotides) that regulate post-transcriptional gene expression in a sequence-specific manner. Since their discovery in C. elegans, microRNAs were found to be expressed in invertebrates and vertebrates, including humans, and many show highly-conservation during evolution (Lagos-Quintana et al., 2001).

MiRNAs regulate gene expression via translational inhibition, mRNA degradation or a combination of both mechanisms (O'Carroll and Schaefer, 2013). In the brain, however, miRNA targeting is frequently not associated with reduced mRNA levels of targets (Klein et al., 2007). Almost 50% of all identified miRNAs are expressed in the mammalian brain and there is significant cell- and region-specific distribution reflecting roles in gene expression that direct the functional specialization of neurons and the morphological responses that are required to adapt to their continuously changing activity state (Siegel et al., 2011; O'Carroll and Schaefer, 2013). MiRNAs and their biogenesis components display select localization within neurons, with significant enrichment in dendrites, enabling local, activitydependent miRNA regulation of protein levels (Lugli et al., 2005, 2008; O'Carroll and Schaefer, 2013). Recent work demonstrated that certain pre-miRs have localization signals which translocate them to the synaptic sites where they are processed into mature miRNA (Bicker et al., 2013).

Most miRNA genes are expressed as a cluster within a single poly-cistronic transcript (Baskerville and Bartel, 2005) and transcribed by RNA polymerase II, resulting in the primary sequence (Cai et al., 2004; Lee et al., 2004). In the nucleus the primary sequence is recognized by the nuclear microprocessor complex containing Drosha and DGCR8 (DiGeorge syndrome critical region 8) which generate the precursor-miRNA (pre-miR) of ∼60–70 nt length (Lee et al., 2003). Next, the precursor is exported into the cytoplasm by Exportin-5 in a GTP-dependent manner (Bohnsack et al., 2004). Once in the cytoplasm, microR-NAs are further processed by Dicer to a single-strand RNA mature form (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Finally, a single strand of the mature miRNA is selected and loaded into the RISC complex and bound to the members of the Argonaute (Ago) protein family. This miRNAbound-RISC complex is the functional and active form of the miRNA which then targets the mRNA. Binding of a 7–8 nt seed region, usually within the 3′UTR of the mRNA, results in either inhibition of the translation or mRNA degradation. This process requires the activation of de-cap enzymes and removal of the poly-A tail (Kwak and Tomari, 2012).

Several studies have shown the role of miRNAs in the brain by using mainly genetic tools in mice, including conditional or full deletion of biogenesis enzymes in the microRNA pathway. Deletion of DGCR8, which affects the production of the precursor microRNA, results in a reduction in brain size and loss of inhibitory synaptic neurotransmission (Babiarz et al., 2011; Hsu et al., 2012). Conditional deletion of Drosha in neural progenitors did not affect neurogenesis in the developing brain, but did affect differentiation and migration of neurons (Knuckles et al., 2012). Deletion of Dicer from neurons produces severe brain abnormalities, including microencephaly and defects in dendritic arborization in cortex and hippocampus (Davis et al., 2008; De Pietri Tonelli et al., 2008; Babiarz et al., 2011; Dorval et al., 2012). Mice lacking Dicer in astrocytes develop spontaneous seizures and suffer from increased premature mortality (Tao et al., 2011). These data imply that the miRNA biogenesis system is essential for brain development and function. Surprisingly, one study reported that specific deletion of Dicer in the adult mouse forebrain transiently enhanced learning and memory, although these animals later displayed degeneration of neurons in the cortex and hippocampus (Konopka et al., 2010).

Analysis of Argonaute (Ago1–4) proteins, main components of the RISC complex, has given more diverse results (Burroughs et al., 2011). Ago-2 is the most abundant expressed member in various tissues including the brain (Liu et al., 2004) and critical for miRNA-mediated repression of mRNAs (Czech and Hannon, 2011). Deficiency in Ago-2 results in death of mice during early embryogenesis or mid-gestation (Morita et al., 2007). This reflects not only the essential role of Ago2 in embryonic development but perhaps an effect of impaired microRNA generation (Morita et al., 2007). In contrast, studies in conditional mutants showed individual deficiencies in Ago 1, 3, and 4 does not produce obvious effects in mice, suggesting a redundancy among Ago family members (O'Carroll et al., 2007).

# microRNA in Ischemia Preconditioning

Three different publications show the regulation of microRNAs after an ischemic preconditioning stimulus in parallel (**Table 1**) (Dharap and Vemuganti, 2010; Lee et al., 2010; Lusardi et al., 2010).

Lusardi et al., using a brief ischemic stimulus in mice, showed that preconditioning at the same time up-regulated and downregulated different microRNAs in the mouse cortex (Lusardi et al., 2010). A common target-gene of the down-regulated microRNAs was the transcriptional regulator MeCP2 (methyl CpG binding protein 2) (Lusardi et al., 2010). Similar results were found when hypertension was used as preconditioning stimulus in rats, where multiple down-regulated microRNAs target MeCP2 (Dharap and Vemuganti, 2010). MeCP2 was first described as a potent transcriptional repressor (Nan, 1998). Mutations in MeCP2 cause Rett syndrome, mental retardation, Angelman syndrome, and autism (Hite et al., 2009; Gonzales and LaSalle, 2010). The up-regulation of MeCP2 via microRNAs corroborated the general gene-repression in tolerance reported before (Lusardi et al., 2010), as increase levels of MeCP2 resulted in a down-regulation of the transcriptome.

In contrast to these studies, Lee et al. showed that neuroprotection achieved by brief ischemic preconditioning stimuli in mice could be due to a family member of miR-200 which targets PHD2 (prolyl hydroxylate 2), which in turn has been involved in the regulation of HIF1α after ischemia (Lee et al., 2010). Upregulation of miR-200 shortly after the preconditioning stimulus reduced the levels of PHD2 and increased HIF1α. These results were corroborated in vitro, where the up-regulation of miR-200 using mimic decreased levels of PHD2 (Lee et al., 2010). Supporting these results, transgenic mice lacking HIF1α exposed to hypoxic preconditioning presented less neuroprotection than control mice after an hypoxia-ischemic insult (Sheldon et al., 2014).

More recently, a model of hibernation, a natural model of tolerance to ischemia, was used to study the role of microRNAs during neuroprotection in the brain (Lee et al., 2012). During torpor stages, hibernating animals lower their energy consumption, blood flow, and body temperature to an otherwise lethal level which is similar to levels during an ischemic insult (Lee et al., 2012). Microarray analysis from ground squirrel brains were performed during the active and the torpor phase and 405 microRNAs were different between both stages, with the family of miR-200 being the most representative, consistent with Lee et al. (2010). In this study the authors suggest that miR-200 family could regulate members of the SUMO family which is involved in the ubiquitin regulation and protein degradation, thereby explaining the reduction in protein levels during tolerance (Lee et al., 2012).

# microRNA in Epileptic Preconditioning

Similar to ischemia, several studies analyzed the role of microRNAs after a preconditioning-seizure stimuli (**Table 1** and Hatazaki et al., 2007; Kretschmann et al., 2015).

McKiernan and colleagues by using low dose of systemic KA in mice showed a de-regulation of microRNA after epileptic preconditioning. Here, a general up-regulation of microRNAs was the main response in preconditioned mice compared to control mice (McKiernan et al., 2012). Whereas 9 microRNAs were uniquely present in the preconditioning group, 39 microRNAs showed increase and 6 reduced levels in the pre-conditioning group. From the up-regulated microRNAs, 25 of the 39 were significantly regulated (McKiernan et al., 2012). Between the upregulated microRNA group, the highest levels were found for miR-184. Inhibition of miR-184 in vivo resulted in neuronal death after a normally non-damaging preconditional stimulus (McKiernan et al., 2012) and an increase in neurodegeneration during the tolerant state after status epilepticus (damaging injury stimulation). Together, these results show a neuroprotective role of miR-184 during preconditioning (McKiernan et al., 2012).

Similar results have been found in acute seizure models not associated with neuronal death (Kretschmann et al., 2015). Kretschman and colleagues showed that 6 h after the induction of a single seizure via electrical stimulation up-regulation of microRNAs was the main response. 146 microRNAs were deregulated, with almost 140 microRNAs being up-regulated and only few of them down-regulated (Kretschmann et al., 2015).

In a more detailed study, the expression levels of microR-NAs were analyzed after electroshock stimulation, showing that microRNA levels follow three distinct patterns after non-damage neuronal activity (Eacker et al., 2011). MicroRNA regulation can be clustered in three distinct groups: Class 1, microRNAs which progressively increase their expression levels after electrical stimulation, including miR-134; Class 2, microRNAs which are strongly up-regulated in the first hours after stimulation with levels decreasing back to normal rapidly; Class 3, microRNAs that increase initially but decrease at a later time-point (12 or 24 h after the stimulation) (Eacker et al., 2011). This study shows the specific response of microRNAs after brain stimulation, and its possible role in the different tolerant-stages.

# Targeting microRNAs on Brain

Several approaches for manipulating miRNA levels in neuronal cells in vitro and in vivo have been described, including recent applications of miRNA antisense oligonucleotides, miRNA gene knockout and miRNA sponges in neuronal cells. The efficiency of microRNAs inhibitors has been reported several times, inhibition of miR184 show a loss in the preconditioning neuroprotection without any toxicity (McKiernan et al., 2012). In contrast, no many approaches have shown over-expression of microRNAs in the brain, possible related with the toxic effect increase levels of RNA in the brain. The study of new drugs which target microR-NAs in the central nervous system will be one of the main fields of future investigations.

# Conclusions

Studying the molecular mechanism underlying the innate neuroprotection can help to understand how the brain protects itself against a damaging insult. Analysis of tolerant states

# References

has shown that a general down-regulation of genes is the main response in the brain, independently of the original insult, ischemic, or seizures. MicroRNAs are master regulators of gene-expression, its regulation after the preconditioning stimulus could be causative of the general suppression of geneexpression and being a good candidate for future target-therapy. Still several questions are remaining: Can we regulate microR-NAs in the brain? Systemic administration of microRNA mimics or microRNA inhibitors will be necessary. Can we regulate microRNA expression in a time dependent manner? Tolerant is a time-dependent effect which only last for several days, a longer inhibition or over-expression of microRNAs could be toxic for the brain.

# Acknowledgments

The author would like to thank Dr. Tobias Engel and Prof. David C. Henshall for critical reading of the manuscript. This work was supported by grants from the Science Foundation Ireland (13/SIRG/2114) to EJ.

humans: the role of comorbidities. Dis. Model. Mech. 7, 1321–1333. doi: 10.1242/dmm.016741

**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.

Copyright © 2015 Jimenez-Mateos. 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.

# Ionic homeostasis in brain conditioning

Ornella Cuomo<sup>1</sup> , Antonio Vinciguerra<sup>1</sup> , Pierpaolo Cerullo<sup>1</sup> , Serenella Anzilotti <sup>2</sup> , Paola Brancaccio<sup>1</sup> , Leonilda Bilo<sup>3</sup> , Antonella Scorziello<sup>1</sup> , Pasquale Molinaro<sup>1</sup> , Gianfranco Di Renzo<sup>1</sup> and Giuseppe Pignataro<sup>1</sup> \*

*<sup>1</sup> Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, Federico II University of Naples, Naples, Italy, <sup>2</sup> SDN Istituto di Ricerca Diagnostica e Nucleare, Naples, Italy, <sup>3</sup> Division of Neurology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, Federico II University of Naples, Naples, Italy*

#### Edited by:

*Mark P. Burns, Georgetown University Medical Center, USA*

#### Reviewed by:

*Zhihui Yang, University of Florida, USA Patrick A. Forcelli, Georgetown University, USA*

#### \*Correspondence:

*Giuseppe Pignataro, Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Via Pansini 5, 80131 Naples, Italy<EMAIL_ADDRESS>
#### Specialty section:

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

Received: *26 March 2015* Accepted: *23 July 2015* Published: *10 August 2015*

#### Citation:

*Cuomo O, Vinciguerra A, Cerullo P, Anzilotti S, Brancaccio P, Bilo L, Scorziello A, Molinaro P, Di Renzo G and Pignataro G (2015) Ionic homeostasis in brain conditioning. Front. Neurosci. 9:277. doi: 10.3389/fnins.2015.00277* Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1–3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na+/Ca2<sup>+</sup> exchangers (NCX), the plasma membrane Ca2+-ATPase (PMCA), the Na+/H<sup>+</sup> exchange (NHE), the Na+/K+/2Cl<sup>−</sup> cotransport (NKCC) and the acid-sensing cation channels (ASIC). Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage.

Keywords: transporters, ionic exchangers, stroke, preconditioning/post-conditioning, brain ischemia

# Ischemic Preconditioning

The concept of ischemic preconditioning was introduced first in the heart (Murry et al., 1986) and later on in the brain (Murry et al., 1986; Schurr et al., 1986). Ischemic preconditioning is neuroprotective strategy in which a subliminal stimulus is applied before a subsequent harmful ischemia (Kirino, 2002; Dirnagl et al., 2003; Gidday, 2006). The state of tolerance mediated by this strategy is composed of two phases of neuroprotection. The first phase, named rapid or acute preconditioning (Perez-Pinzon et al., 1997), starts 3–5 min after the preconditioning stimulus and ends 1 h later and is due to rapid post-translational protein modifications (Barone et al., 1998; Meller et al., 2006). The second phase, named delayed preconditioning (Perez-Pinzon et al., 1997), starts 2–3 days after preconditioning and ends 1 week later and mainly involves de novo protein synthesis (Barone et al., 1998; Dirnagl et al., 2003; Meller et al., 2005).

# Mechanisms Involved in Ischemic Preconditioning Neuroprotection

The molecular mechanisms underlying ischemic tolerance are not yet fully understood because of its extreme complexity, involving many signaling pathways and alterations in gene expression. Several studies have investigated the signaling cascades and key molecules both in in vitro and in vivo models of ischemic preconditioning. Most of the stressors induce both rapid and delayed tolerance. As recently reviewed (Obrenovitch, 2008), the features of ischemic tolerance partially are similar to naturally occurring adaptive mechanisms, including at cellular levels:

# Ischemic Post-conditioning

Unlike ischemic preconditioning, ischemic post-conditioning is a relatively novel neuroprotective strategy (Zhao et al., 2006; Pignataro et al., 2008). Rapid revascularization of the occluded vessels represent one of the most effective approaches currently used for the treatment of acute ischemic stroke. However, during the early reperfusion phase an increase in reactive oxygen species and intracellular free Ca2<sup>+</sup> may occur, thus leading to additional injury (Kuroda and Siesjö, 1997). Ischemic post-conditioning is realized by applying brief interruptions of reperfusion after ischemia. That a protocol of cycles of brief reperfusion followed by re-occlusion was able to reduce the ischemic damage was first demonstrated during cardiac ischemia (Zhao et al., 2003). More recently, it has been demonstrated that ischemic postconditioning induces neuroprotection also in a rat hippocampal slice model of cerebral ischemia (Scartabelli et al., 2008), in rodent models of spinal cord injury (Jiang et al., 2006), and in models of focal (Zhao et al., 2006; Pignataro et al., 2008, 2013b; Xing et al., 2008), and global ischemia (Pateliya et al., 2008; Wang et al., 2008).

## Mechanisms Involved in Ischemic Post-conditioning Neuroprotection

At present a few information are available about the mechanisms involved in the neuroprotection mediated by ischemic postconditioning. Since post-conditioning is applied after the ischemic injury, the changes observed in the brain have to be ascribed to the combined effects of post-conditioning and stroke, rather than the effect of the post-conditioning only. Thus, it is difficult to sunder the protective mechanisms of postconditioning from the consequences of its protection. However, up until now, many mechanisms have been suggested (Zhao, 2009), such as:

1. Reduction of hyperemia consequent to the reperfusion, that may be accompanied other toxic events, such as the production of free radicals, loss of blood–brain barrier integrity, and activation of inflammation cascade (Schaller and Graf, 2004).

# Brain Ionic Transporters during Ischemic Conditioning

Ischemic brain conditioning activates intracellular biological responses to a potential lethal insult; therefore it is predictable that organs reinforce their tolerance when exposed to a sublethal insult prior or after a harmful episode of ischemia by increasing energy metabolism or delaying anoxic depolarization after the onset of the subsequent ischemic insult (Stenzel-Poore et al., 2003; Pignataro et al., 2008, 2009). Indeed, an impairment in the activity of voltage-gated potassium channels has been observed in cortical neurons exposed to a brief non-injurious oxygen and glucose deprivation (OGD). Moreover, it has been demonstrated that ischemic conditioning prevented the inhibition of Na+/K+-ATPase activity after brain ischemia in hippocampal and cortical neurons of rats subjected to global forebrain ischemia. Instead, in vivo experiments in gerbils showed that both Ca2+-ATPase activity and mitochondrial calcium internalization increased in CA1 hippocampal neurons exposed to preconditioning (Ohta et al., 1996). Accordingly, in hippocampal neurons obtained from preconditioned gerbils [Ca2+]<sup>i</sup> increased after anoxic and aglycemic episodes whereas this increase was inhibited in ischemic-tolerant animals (Shimazaki et al., 1998). The molecular mechanisms of this phenomenon are still under investigation. An explanation may be the increase in the expression of the plasma membrane calcium ATPase 1 (PMCA-1) as recently demonstrated (Kato et al., 2005). Moreover, many evidence support the idea that other transporters are involved in the protection mediated by brain conditioning. In this regard, a key role is exerted by the sodium calcium exchanger (NCX), in fact its expression was reduced during cerebral ischemia (Pignataro et al., 2004; Boscia et al., 2006). More important, NCX downregulation was prevented when rats were subjected either to ischemic preconditioning or post-conditioning (Pignataro et al., 2013a). In addition Na+/H<sup>+</sup> exchanger and Na+/K+/2Cl<sup>−</sup> cotransporter are critical ion transporters in the context of cerebral ischemia, as they contribute to the regulation of intracellular pH and cell volume (Pedersen, 2006). Finally, acid-sensing cation channels (ASIC) have also been studied during ischemic conditions since they represent key targets in many different aspects of acidosis, a condition that occurs during ischemic stroke. Brain preconditioning as an adaptive response to stroke requires in particular new protein transcription and translation; giving that HIF-1–dependent transcription is being clearly common in activating pathways of several exchangers/transporters/ionic channels involved in stroke, scientific community has assigned a key role to HIF-1α, as a master transcriptional regulator of cellular and developmental response to hypoxia. In effect stable HIF-1 can bind to its heterodimeric partner HIF-1β, and together these proteins, once stabilized during hypoxicischemic conditions, can act in the nucleus to transactivate genes involved in adaptation to hypoxic-ischemic stress. HIF represents the "hyphen" that definitely binds to each other all the different mechanisms converging on the same signaling cascade, responsible for the homeostatic regulation in protected brain (Ratan et al., 2004). In the light of these premises, the present article will review the main role of protein transporters and exchangers controlling ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning, with particular regards to the Na+/Ca2<sup>+</sup> exchangers (NCX), the plasma membrane Ca2+-ATPase (PMCA), the Na+/H<sup>+</sup> exchange (NHE), the Na+/K+/2Cl<sup>−</sup> cotransport (NKCC) and the acid-sensing cation channels (ASIC).

# Role of Na+/H<sup>+</sup> Exchange (NHE) and Na+/K+/2Cl<sup>−</sup> Cotransport (NKCC) In Brain Conditioning

The ubiquitous plasma membrane Na+/H<sup>+</sup> exchanger is protein highly conserved across vertebrate species and characterized as a major membrane transporter involved in the regulation of cellular pH and volume in the nephron of the kidney. Specifically it is present in the intercalary cells of the collecting duct (Pedersen, 2006). The Na+/K+/2Cl<sup>−</sup> cotransporter (NKCC) is a protein that aids in the active transport of sodium, potassium, and chloride into and out of cells; it is widely distributed throughout the body and it has important functions in organs that secrete fluids. NKCC2 is found specifically in the kidney,

post-conditioning.

where it serves to extract sodium, potassium, and chloride from the urine so that they can be reabsorbed into the blood (Haas, 1994).

Na+/H<sup>+</sup> exchanger and Na+/K+/2Cl<sup>−</sup> cotransporter are critical ion transporters in the context of cerebral ischemia, as they contribute to the regulation of intracellular pH and cell volume (Pedersen, 2006). In particular, while the mammalian NHE is an integral membrane transport protein that mediates an electroneutral 1:1 exchange of intracellular H<sup>+</sup> for extracellular Na<sup>+</sup> (Xia et al., 2003), the electroneutral NKCC contransporter mediates the influx of Na+, K+, and Cl<sup>−</sup> with a stoichiometry of 1Na+:1K+:2Cl−.

Due to this dual role it is difficult to predict whether a change in the expression and in the activity of these transporters may ameliorate or impair the susceptibility of cells to ischemia; for example, enhanced Na+/H<sup>+</sup> exchange would help to reduce the intracellular pH but, on the other hand, it would promote cell swelling and further Ca2<sup>+</sup> influx through Na+/Ca2<sup>+</sup> exchange. In particular, in cortical astrocyte cultures obtained from Na+/H<sup>+</sup> exchanger isoform 1-deficient NHE1(-/-) mice the lack of NHE1 attenuated the damage induced by conditions mimicking cerebral ischemia in vitro, and this effect was confirmed also in vivo (Kintner et al., 2004). Indeed, the pharmacological blockade of NHE-1 reduces infarct volume in animal models of brain ischemia. In addition, ischemia/reperfusion causes an increase in NHE-1 expression/activity (Ramasamy et al., 1995).

Although NHE activation is essential for the restoration of physiological pH<sup>i</sup> , hyperactivation of NHE1 in neurons, in response to the metabolic acidification associated with an ischemic–hypoxic insult (Vornov et al., 1996; Luo et al., 2005; Hwang et al., 2008; Kersh et al., 2009), disrupts the intracellular ion balance, causing intracellular Na<sup>+</sup> and Ca2<sup>+</sup> overload (Matsumoto et al., 2004), which eventually leads to cell death. Consequently the in vitro and in vivo approach of several studies have demonstrated neuroprotection with NHE inhibitors.

Similar data were obtained with genetic manipulations of NKCC1 in mice and by the inhibition of NKCC1 using bumetanide (Chen and Sun, 2005). NKCC1 is expressed in neurons throughout the brain where it contributes to the maintenance of [Cl−]<sup>i</sup> . Thus, it may affect neuronal excitability by regulating [Cl−]<sup>i</sup> . Expression of NKCC1 protein has also been found in astrocytes and oligodendrocytes (Lenart et al., 2004). The pharmacological inhibition or transgenic ablation of NKCC1 significantly attenuates infarction and swelling after brain ischemia induced by transient middle cerebral artery occlusion (tMCAO) (Chen and Sun, 2005). Therefore, it may be hypothesized that NKCC1 represents an attractive target for conditioning strategies against stroke.

# NHE and NKCC Role during Ischemic Preconditioning

NHE and NKCC role during ischemic preconditioning is still controversial and very little is known about their role in the processes triggered by brain preconditioning, since all the studies were conducted in the heart. According to a first theory, activation of both transporters participates in the protection mediated by ischemic cardiac preconditioning. Indeed, the reduced acidification occurring during ischemia (Schaefer et al., 1995) along with the increase in intracellular sodium following preconditioning is consistent with a modulation of these proteins by preconditioning (Ramasamy et al., 1995). These mechanisms may include the activation of both NKCC and NHE, potentially resulting in reduced intracellular acidosis during ischemia. NKCC regulation is multifactorial, with evidence that the cotransporter is regulated by changes in cell volume (O'Donnell, 1993) and cAMP-dependent (Pewitt et al., 1990; Lytle et al., 1992; Klein, 1993) and non cAMP-dependent protein phosphorylation as well as ionic concentration gradients. Increased inward cotransporter flux during ischemia after preconditioning could be beneficial in protecting the heart through its functional coupling with the Cl−/HCO3<sup>−</sup> exchanger (Anderson et al., 1990). In fact, chloride transported into the cell by the Na+-K+-2Cl<sup>−</sup> cotransporter may exit via Cl−/HCO<sup>−</sup> 3 exchange, thus increasing intracellular HCO3<sup>−</sup> and limiting acidosis.

The primary factors regulating NHE include the intracellular proton concentration and the phosphorylation state. Under baseline conditions, the exchanger contributes to the extracellular/intracellular proton gradient with net outward transport of protons (Piwnica-Worms et al., 1986). Under conditions of ischemia in which protons are generated and intracellular pH decreases, the exchanger is stimulated to increase extrusion of protons, thus increasing the inward transport of sodium ions. However, this hypothesis has been more recently disproved. In fact, it has been highlighted that the above discussed study was carried out in hearts perfused with a medium containing HCO3<sup>−</sup> and the method used to induce acute intracellular acidosis resulted in greater acidosis in preconditioned than in control hearts. For these reasons, the faster initial recovery from acidosis in preconditioned hearts may be a consequence of the lower starting pH<sup>i</sup> . Indeed, de Albuquerque et al. (1995) recently showed that, in a similar condition, the rate of pH<sup>i</sup> recovery is similar in control and preconditioned rat hearts. More recently, it has been demonstrated that NHE activity did not contribute to the cardioprotection mediated by ischemic preconditioning. Indeed, inhibition of Na+/H<sup>+</sup> exchanger isoform-1 (NHE1) is protective in adult myocardium in a model of ischemia followed by reperfusion; however, the effect is unclear in immature myocardium (Cun et al., 2007). More important, the administration of HOE, a NHE inhibitor, did not prevent the protection induced by preconditioning, thus suggesting that NHE inhibition during the prolonged ischemic period may enhance the protection afforded by preconditioning (Shipolini et al., 1997). Accordingly, another group demonstrated that both NHE inhibition and ischemic preconditioning eliminated the increase in intramyocyte Na<sup>+</sup> content that otherwise occurred with cardioplegic arrest and reperfusion in a porcine model. Because their mechanisms are distinct, the authors proposed that an additive beneficial effect against ischemia-reperfusion injury can be achieved by using NHE inhibition together with a preconditioning stimulus as prereperfusion therapy (Goldberg et al., 2002).

# NHE and NKCC Role during Ischemic Post-conditioning

Up until now, there is few information about the role of NHE1 exchanger in brain post-conditioning since many studies have demonstrated its neurodegenerative role in brain ischemia upon activation. According to these data, the ischemic neuroprotection induced by post-conditioning may be associated with the downregulation of NHE1 expression or activity. These hypothesis are supported by few experimental evidences. In this regard, some information were obtained in the heart where it has been demonstrated the role of NHE in a delayed recovery of pH<sup>i</sup> after heart post-conditioning. Indeed, it has been suggested that prolongation of acidosis during reperfusion is important for the post-conditioning-mediated neuroprotection. Inserte et al. demonstrated that such post-conditioning protocols that significantly prolong pH<sup>i</sup> recovery during initial reperfusion are protective for the heart. Accordingly, there is a narrow inverse correlation between the level of the delay and the extent of cell death, demonstrating the key role of the delay of intracellular acidosis in the protection mediated by post-conditioning (Inserte et al., 2009). The normalization of pH<sup>i</sup> during reperfusion is mediated by the combined action of different transport systems, including Na+/H+-exchanger, Na+/HCO3<sup>−</sup> symport, and H+ coupled lactate efflux (Vandenberg et al., 1993). These systems appear to be mainly redundant, since inhibition of one of them does not result in significant delay in pH<sup>i</sup> recovery. For instance, inhibition of Na+/H+-exchanger by selective agents administered at the onset of reperfusion has a very small effect on pH<sup>i</sup> recovery (Docherty et al., 1997; Ten Hove and Van Echteld, 2004). By contrast, simultaneous inhibition of Na+/H+ exchanger and Na+/HCO3<sup>−</sup> symport delay normalization of pH<sup>i</sup> (Docherty et al., 1997; Schäfer et al., 2000; Inserte et al., 2008).

The concept that pH<sup>i</sup> correction was correlated to the washout of lactate, H<sup>+</sup> and CO<sup>2</sup> was supported by the strong correlation between the delay in pH<sup>i</sup> recovery and the levels of lactate measured in the coronary effluent (blood outflow through coronaries) during the first 2 min of reperfusion (Vandenberg et al., 1993; Inserte et al., 2008). Thus, reduced catabolite washout caused an attenuation of the transmembrane H<sup>+</sup> gradient and a decreased activity of Na+/H+-exchanger and Na+/HCO3<sup>−</sup> symport.

The protective effect of prolongation of intracellular acidosis during reperfusion has been solidly demonstrated in different models, including isolated cardiomyocytes (Schäfer et al., 2000) and intact hearts (Kitakaze et al., 1988; Ohashi et al., 1996; Preckel et al., 1998; Inserte et al., 2008). Intriguingly, there is a correlation between delayed acidosis and other cardioprotective pathways during post-conditioning such as Reperfusion Injury Salvage Kinases (RISK) (Fujita et al., 2007). According to this theory, alkalotic buffer reduced Akt and ERK phosporylation in post-conditioned myocardium.

Regarding the role of Na+-dependent chloride transporter (NKCC) during brain post-conditioning few information are available. In a recent work it has been demonstrated that in hippocampal neurons exposed to hypoxia conditioning mimiking an in vitro post-conditioning, NKCC1 is strongly activated through binding with STE20/SPS1-related proline/alanine-rich kinase (SPAK) (Yang et al., 2013). This finding could be considered as starting point for future studies.

# Role of Acid-sensing Cation Channels (ASIC) in Brain Conditioning

Acid-Sensing Ion Channels (ASICs) are neuronal voltageinsensitive cationic channels activated by extracellular protons. ASIC proteins are a subfamily of the ENaC/Deg superfamily of ion channels. ASICs are potential drug targets for treating a wide variety of conditions linked to both the CNS and PNS. Acid-sensing cation channels (ASIC) have been extensively studied both in physiological and in pathological conditions for their wide distribution in the nervous system and since they represent key targets in many different aspects of acidosis, a condition that occurs during ischemic stroke, as previously analyzed (Waldmann et al., 1997; Wemmie et al., 2006). Until now, six ASIC subunit proteins, encoded by four genes, have been identified: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4; among these, the subtypes 1a, 2a, and 2b are expressed in neurons of the CNS. All ASICs belong to the degenerin/epithelial Na<sup>+</sup> channel (DEG/ENaC) superfamily, which are Na+- selective cation channels sensitive to amiloride (Crowell and Kaufmann, 1961; Sutherland et al., 2000). Blockade of ASIC1a and deletion of the ASIC1 gene rescued neurons from ischemic cell death. In CNS neurons, ASIC1a responds to extracellular pH reduction ranging from 6.9 to 5.0 to generate rapid depolarizing currents (Waldmann et al., 1997), and its activation enhances the probability of action potential initiation (Vukicevic and Kellenberger, 2004). ASIC1a channels are activated at the pH values associated with CNS diseases (ischemia, ∼pH 6.5–6.0; seizure, ∼pH 6.8; AD, ∼pH 6.6). In the case of homomeric ASIC1a channels, acid activation also induces Ca2<sup>+</sup> entry directly through these channels (Crowell and Kaufmann, 1961; Nedergaard et al., 1991; Dietrich and Morad, 2010). In addition, the ASIC-mediated membrane depolarization may facilitate the activation of voltage gated Ca2<sup>+</sup> channels and NMDA receptor-gated channels (Gao et al., 2005), further promoting neuronal excitation and [Ca2+]<sup>i</sup> accumulation.

Acidosis usually occurs during many central nervous system (CNS) diseases. Indeed, ischemic brain pH falls to 6.0 due to the accumulation of lactic acid and to the protons produced by ATP hydrolysis (Nedergaard et al., 1991; Gao et al., 2005). So the reasonable strategy to contrast pH lowering, sustained by ASICs activation, is to block its activity, since acidosis is a common feature of acute neurological conditions such as ischemic stroke, brain trauma, and epileptic seizures (Rehncrona, 1985). It is rational to suggest that restoration of acid–base balance and blockade of the down-stream pathways of acidosis may represent two promising strategies to avoid the consequences of acidosis.

In this direction ASIC involvement in neuroprotection elicited in the brain by pre- and post-conditioning has been investigated (Pignataro et al., 2007).

Indeed, Pignataro and colleagues demonstrated that ASIC1a is downregulated during both preconditioning and postconditioning and that this downregulation was dependent on p-Akt. Particularly, p-Akt is up-regulated during ischemic preand post-conditioning. In fact, during harmful ischemia, Akt is transiently phosphorylated, and then activated for a short time interval after reperfusion (Beg et al., 2008), whereas after brain conditioning the phosphorylation of Akt persists longer, even 24 h later, the same time interval at which an ASIC1a downregulation occurs. Interestingly, selective p-Akt inhibition prevented ASIC1a downregulation thus reverting the conditioning-induced neuroprotection.

That p-Akt is the mediator of the downregulation of ASIC1a during pre- and post-conditioning are supported by previous papers showing that Reperfusion Injury Salvage Kinasesì (RISKs), including Akt (Nedergaard et al., 1991; Xu and Xiong, 2007; Beg et al., 2008; Pignataro et al., 2011a) are activated during conditioning. In conclusion, since reduction of ASIC1a is a key factor in the neuroprotective effect of pre- and post-conditioning, the downregulation of its expression and activity might be a potential strategy to ameliorate the ischemic damage. Besides ASIC1a, ASIC2a has also been linked to ischemic preconditioning neuroprotection. In fact, the upregulation of ASIC2a is associated to the neuroprotective effect of ischemic preconditioning on global brain ischemia (Miao et al., 2010).

# Role of Sodium/Calcium Exchanger (NCX) during Brain Conditioning

The sodium-calcium exchanger (Na+/Ca2<sup>+</sup> exchanger, NCX, or exchange protein) is an antiporter membrane protein that removes calcium from cells. It consists of nine transmembrane segments that can mediate Ca2<sup>+</sup> and Na<sup>+</sup> fluxes across the plasma membrane. Depending on the intracellular concentrations of Ca2+, [Ca2+]<sup>i</sup> , and Na+, [Na+]<sup>i</sup> , NCX can operate either in the forward mode, coupling the uphill extrusion of Ca2<sup>+</sup> to the influx of Na<sup>+</sup> ions, or in the reverse mode, mediating the extrusion of Na<sup>+</sup> and the influx of the Ca2<sup>+</sup> ions (Pignataro et al., 2004). The NCX is considered one of the most important cellular mechanisms for removing Ca2+. The maintenance of Na<sup>+</sup> and Ca2<sup>+</sup> homeostasis, mediated by this exchanger, is a crucial physiological phenomenon in neuronal and non-neuronal cells. NCX countertransports four or three Na<sup>+</sup> ions in change of one Ca2<sup>+</sup> ion (Reeves and Hale, 1984; Fujioka et al., 2000; Kang and Hilgemann, 2004); it is a highcapacity and low-affinity ionic transporter. When intracellular Ca2<sup>+</sup> concentrations ([Ca2+]i) increase this exchanger mediates the entrance of sodium ions and the extrusion of calcium ions in tight dependence of their electrochemical gradient. The way in which the antiporter works (forward or reverse) depends on several factors such as membrane potential, Na<sup>+</sup> and Ca2<sup>+</sup> gradient. Therefore, NCX represents an important actor in the regulation of pathophysiological and physiological responses to increases of [Ca2+]<sup>i</sup> and [Na+]<sup>i</sup> (Haddad and Jiang, 1997; Dirnagl et al., 1999; Annunziato et al., 2004) In particular, three distinct NCX genes, ncx1-3, and several splicing variants have been so far described (Nicoll et al., 1990, 1996). The CNS is the only organ that expresses all three NCX gene products. NCX is particularly expressed in neurons at the level of synapses (Canitano et al., 2002) where, in the course of an action potential, Ca2<sup>+</sup> crosses the plasma membrane. Calcium entry allows the fusion of synaptic vesicles with the plasmamembrane and permits the neurotransmitter release. After that, plasma membrane Ca2<sup>+</sup> ATPase and NCX mediate the extrusion of the residual intracellular Ca2+. In presence of [Ca2+]<sup>i</sup> higher than 500 nM, NCX become the prevalent Ca2<sup>+</sup> extrusion mechanism (Pignataro et al., 2014). At the same time, the deregulation of [Ca2+]<sup>i</sup> and [Na+]<sup>i</sup> homeostasis may mediate the damage of neuronal and glial cells occurring after stroke and other neurodegenerative disorders.

# Na+/Ca2<sup>+</sup> Exchanger in Brain Preconditioning

It is well-known that NCX may play a role in the maintenance of ionic homeostasis during ischemic conditioning (Pignataro et al., 2004, 2012; Molinaro et al., 2013). In this respect, NCX is downregulated during brain ischemia and this downregulation occurs in a manner dependent on the exchanger isoform and on the brain region interested by the damage (Pignataro et al., 2004; Boscia et al., 2006). It has been recently demonstrated that NCX1 and NCX3 isoforms represent two new effectors of the preconditioning-mediated neuroprotection (Pignataro et al., 2012). In fact, while the absence of NCX1 and NCX3 is able to partially prevent the neuroprotection mediated by ischemic preconditioning, the pro-survival factor p-Akt mediated preconditioning-induced brain protection by inducing an upregulation of NCX1 and NCX3. In this field, these results have been obtained in an experimental model of ischemic preconditioning in which preconditioning was mediated in rats by 30 min occlusion of the middle cerebral artery (MCA), followed by a subsequent harmful stimulus of 100 min of MCA occlusion, applied 72 h after the preconditioning stimulus (Pignataro et al., 2012). The correlation between NCX and p-Akt was demonstrated also by confocal fluorescence experiments, that showed a colocalization among NCX1, NCX3, and p-Akt in the temporoparietal cortex of preconditioned rats (Formisano et al., 2008; Pignataro et al., 2012). Moreover, the PI3-K inhibitor, LY294002, reverts the protective outcome elicited by ischemic preconditioning, thus giving further support to the role of p-Akt in this phenomenon (Endo et al., 2006; Pignataro et al., 2008). The overexpression p-Akt-dependent of NCX1 and NCX3 elicited by brain preconditioning may be considered a response of cells to overcome the deregulation of ionic homeostasis occurring in the brain under anoxic conditions. On the other hand, p-Akt is not the only transducer able to activate NCX1 and NCX3. Indeed, Valsecchi and colleagues underlineed that after ischemic preconditioning, hypoxia-inducible factor, HIF-1α, is strongly increased and mediated the increased expression of NCX1 expression, thus contributing to brain protection induced by ischemic preconditioning (Valsecchi et al., 2011) and suggesting that ncx1 gene represents a new HIF-1 target (Valsecchi et al., 2011). Summarizing, NCX1 and NCX3 overexpression may be considered as a mechanism that preconditioning activates in neurons and glial cellsin order to overcome the loss of ionic homeostasis caused by harmful ischemia. Interestingly, the activation of these mechanisms are long lasting, since NCX1 and NCX3 upregulation is still present 72 h after preconditioning induction, thus suggesting that these two isoforms can be included in the list of effectors able to mediate the so-called delayed preconditioning (Pignataro et al., 2013a).

# Na+/Ca2<sup>+</sup> Exchanger in Brain Post-conditioning

Regarding NCX role during brain post-conditioning, NCX3 has been confirmed as a molecular effector involved in the neuroprotection (Pignataro et al., 2011b). In particular, interesting results have been obtained in an experimental model of ischemic post-conditioning, performed in the laboratory of Pignataro and colleagues by subjecting adult male rats to 10 min of MCAO 10 min after 100 min of MCAO. Results showed that p-Akt expression after post-conditioning increased and timely mirrors the increase in NCX3 expression. On the other hand, NCX3 silencing reverted the post-conditioning-mediated neuroprotection and the selective inhibition of p-Akt prevented NCX3 upregulation and the neuroprotection (Pignataro et al., 2011b). NCX3 overexpression during post-conditioning may contribute to counteract the ionic homeostasis dysregulation. By contrast NCX1 silencing did not revert the post-conditioninginduced neuroprotection, thus showing that NCX1 does not play a relevant role during post-conditioning. This result can be explained by the observation that unlike NCX1 and NCX2, the isoform NCX3 may be still operative during ATP depletion (Linck et al., 1998; Secondo et al., 2007). In addition, the differences in NCX1 and NCX3 promoters render NCX3 a better target for the prosurvival kinase cAMP response elementbinding protein (CREB), an Akt downstream player (Gabellini et al., 2003). After post-conditioning, the phosphorylation of Akt is present longer (Pignataro et al., 2008) and timely parallels the intervals of NCX3 upregulation. Furthermore, confocal experiments in the temporoparietal confirmed that the increased expression of these two proteins after post-conditioning occurs in the same cells. These considerations, beside showing that p-Akt palys a key role in neuroprotection, more important suggest NCX3 as one of the additional p-Akt effectors involved in the neuroprotective effect of ischemic post-conditioning.

# Role of Plasma Membrane Ca2<sup>+</sup> ATPase (PMCA) in Brain Conditioning

The plasma membrane Ca2<sup>+</sup> ATPase (PMCA) is a transport protein in the plasma membrane of cells that functions removing calcium (Ca2+) from the cell; so it is also an important regulator of the calcium concentration in the extracellular space. This protein is the only plasmamembrane high affinity Ca2<sup>+</sup> transporting system. It removes Ca2<sup>+</sup> from the cytosol in all eukaryotic cells, and for most of them it is the only Ca2<sup>+</sup> exporting system together with Na+/Ca2<sup>+</sup> exchanger which represent the principal Ca2<sup>+</sup> exchanger in heart cells and neurons (Jensen et al., 1993). The high Ca2<sup>+</sup> affinity of the pump, however renders it the only system capable of finetuning its exchanges with the external ambient (Brini et al., 2013).

Conversely to other plasma membrane buffering systems, such as NCX and ATPases, plasma membrane Ca2<sup>+</sup> -ATPase may export intracellular Ca2<sup>+</sup> even with relatively lower transport capacity during prolonged membrane hyperpolarization and

# References

during conditions of increased intracellular Na<sup>+</sup> concentrations, as occurred under ischemic conditions. Ohta et al. (1996) suggested that ischemia-tolerant CA1 neurons, in a model of bilateral common carotid arteries occlusion in gerbils, export more efficiently a larger amount of Ca2<sup>+</sup> (Kato et al., 2005).

Moreover, in gerbils, ischemic preconditioning by preexposure to 2-min ischemia is extremely effective in protecting against ischemic cell death since there is a modification of the electrophysiological properties of CA1 neurons (Kawai et al., 1998; Shimazaki et al., 2000). In this experimental conditions, expression of PMCA1 is increased in the hippocampus following 2-min ischemia (Kato et al., 2005). The same authors evaluated PMCA1 expression also in a model of tolerance induced by a low dose treatment with 3-Nitro Propionic Acid (3-NPA). They observed an increase in the expression of Bcl-2 in the hippocampus in both the models of preconditioning. However, PMCA1 increased just in the 2-min ischemia model and not in the 3-NPA model, thus suggesting that it plays an important but not essential role in the enhancement of ischemic tolerance (Kato et al., 2005).

# Conclusions

The identification of the players involved in the control of ionic homeostasis during pre and post-conditioning should provide more direct opportunities for translational neuroprotection trials. Indeed, the present review, summarizing the specific effect of the plasmamembrane proteins involved in the maintenance of cellular ionic concentration, would provide information of which transporters needed to be activated or inhibited in order to protect the brain by the ischemic injury.

NCX1, NCX2, and NCX3 and their related proteins in the central nervous system. Ann. N.Y. Acad. Sci. 976, 394–404. doi: 10.1111/j.1749-6632.2002.tb0 4766.x

postconditioning and identification of potential candidates for stroke therapy. FEBS J. 276, 46–57. doi: 10.1111/j.1742-4658.2008.06769.x

**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 Cuomo, Vinciguerra, Cerullo, Anzilotti, Brancaccio, Bilo, Scorziello, Molinaro, Di Renzo and Pignataro. 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.

# **Novel cellular mechanisms for neuroprotection in ischemic preconditioning: a view from inside organelles**

#### *Maria Josè Sisalli <sup>1</sup> , Lucio Annunziato1,2 and Antonella Scorziello<sup>1</sup> \**

*<sup>1</sup> Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Science, School of Medicine, Federico II University of Naples, Naples, Italy, <sup>2</sup> Fondazione IRCSS SDN, Naples, Italy*

Ischemic preconditioning represents an important adaptation mechanism of CNS, which

#### *Edited by:*

*Alexej Verkhratsky, University of Manchester, UK*

### *Reviewed by:*

*Dandan Sun, University of Pittsburgh, USA Zhihui Yang, University of Florida, USA*

#### *\*Correspondence:*

*Antonella Scorziello, Department of Neuroscience, Reproductive and Odontostomatological Science, School of Medicine, Federico II University of Naples, Via S. Pansini 5, Naples 80131, Italy<EMAIL_ADDRESS>
#### *Specialty section:*

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

> *Received: 02 February 2015 Accepted: 05 May 2015 Published: 26 May 2015*

#### *Citation:*

*Sisalli MJ, Annunziato L and Scorziello A (2015) Novel cellular mechanisms for neuroprotection in ischemic preconditioning: a view from inside organelles. Front. Neurol. 6:115. doi: 10.3389/fneur.2015.00115* results in its increased tolerance to the lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and not yet completely clarified. In the last 10 years, great attention has been devoted to unravel the intracellular pathways activated by preconditioning and responsible for the establishing of the tolerant phenotype. Indeed, recent papers have been published supporting the hypothesis that mitochondria might act as master regulators of preconditioning-triggered endogenous neuroprotection due to their ability to control cytosolic calcium homeostasis. More interestingly, the demonstration that functional alterations in the ability of mitochondria and endoplasmic reticulum (ER) managing calcium homeostasis during ischemia, opened a new line of research focused to the role played by mitochondria and ER cross-talk in the pathogenesis of cerebral ischemia in order to identify new molecular mechanisms involved in the ischemic tolerance. In line with these findings and considering that the expression of the three isoforms of the sodium calcium exchanger (NCX), NCX1, NCX2, and NCX3, mainly responsible for the regulation of Ca<sup>2</sup><sup>+</sup> homeostasis, was reduced during cerebral ischemia, it was investigated whether these proteins might play a role in neuroprotection induced by ischemic tolerance. In this review, evidence supporting the involvement of ER and mitochondria interaction within the preconditioning paradigm will be provided. In particular, the key role played by NCXs in the regulation of Ca<sup>2</sup><sup>+</sup>-homeostasis at the different subcellular compartments will be discussed as new molecular mechanism proposed for the establishing of ischemic tolerant phenotype.

**Keywords: ischemic preconditioning, sodium calcium exchanger, mitochondria, neurons, calcium**

# **Introduction**

Cerebral ischemia is a multifactorial and complex disease (1, 2). Indeed, the intracellular events activated by the loss of perfusion of the brain and responsible for neuronal damage range from impairment of intracellular homeostasis to mitochondrial dysfunction and free radical production (3–5). The complexity of these events explains the great discrepancy between the frequency of cerebral ischemic accidents and the lack of effective treatments able to inhibit or slow neuronal demise following the ischemic insult. Hence, the urgent need to identify new potential targets for the development of innovative therapeutic strategies able to defend the ischemic brain.

On these premises, in the recent years, the attention of the researchers focused on ischemic tolerance a phenomenon, also known as ischemic preconditioning (IPC), which consists of a sub-lethal anoxic insult that makes the tissue in which it occurs more resistant to a subsequent and potentially lethal ischemia (6–11). The relevance of this phenomenon is to correlate to the study of the endogenous mechanisms activated in neurons to allow cell survival after a sub-lethal ischemic stimulus. By this way, it is possible to identify new molecular targets useful to develop alternative therapeutic strategies to treat the ischemic disease. The great interest in the cerebral IPC and in the tolerance evoked by itself also comes from the similarity of this phenomenon with those clinical situations occurring in the human brain. Indeed, it is well known that transient ischemic attacks (TIAs) do not cause structural damage but appear to protect brain against a subsequent "stroke" (12, 13).

Therefore, IPC or ischemic tolerance of the brain lie in a natural adaptive process that can be mimicked by a variety of sub-lethal insults, such as transient hypoxia, spreading depression, oxidative stress, hyperthermia, or heat shock, and that increases the tissue tolerance to a subsequent, potentially lethal ischemia. This adaptive cytoprotection is a fundamental property of living cells, which allows them to survive to the exposure to potentially recurrent stressors. This phenomenon was clearly identified in the heart by Murry et al. (14) as preconditioning, or subsequently as ischemic tolerance, and in 1990 it was described also in the brain by Kitagawa et al. (15). Since then, it immediately attracted the interest of clinical and basic neuroscientists for several reasons. First, this biological process became widely recognized as a pertinent and effective experimental instrument to understand how the brain protects itself against ischemia, thereby providing an innovative approach for the discovery of novel neuroprotective strategies. Second, retrospective case-control studies showed a clinical correlate of the phenomenon discovered experimentally.

# **Molecular Mechanisms Involved in Ischemic Preconditioning**

Among the molecular mechanisms underlining ischemic tolerance, the substantial changes in gene expression play a crucial role, suggesting that preconditioning is able to stimulate a genomic reprograming of cells which in turn is responsible for the cytoprotection and cellular survival described in those tissues interested by the ischemic event (16, 17). The effects in the cell genome, occurring after IPC, represent the signature of the complex interplay of multiple signaling pathways. It is interesting to underline that they are highly specialized pathways in different cell types of the brain and concur to the cellular and systemic response activated in the tissue to combat the noxious stimulus. Indeed, it has been reported that hundreds of genes undergo upregulation or downregulation in response to IPC stimuli (18–20), and that these changes in gene expression differ between the harmful ischemic insult and the sub-lethal IPC. In particular, preconditioning seems to attenuate the response to ischemia (19), and the tolerance established modifies the expression of genes involved in the suppression of metabolic pathways, in the control of immune responses, in the modulation of ion-channel activity, and in the changes of blood coagulation parameters (19).

As recently reported, three phases, temporally consecutive of the preconditioning phenomenon have been identified as induction, transduction, and tolerance.

# **Induction and transduction mechanisms of tolerance**

The induction of tolerance requires that the preconditioning stimulus has to be recognized by molecular sensors as a sign of a more severe phenomenon that will occur. This implicated the identification of numerous types of sensors, including neurotransmitters, neuromodulators, cytokines, and toll-like receptors (21) as well as ion channels and redox-sensitive enzymes (22– 24) which in turn will activate subsequent transduction pathways responsible for initiating the adaptive response. The activation of the transduction phase also implies the involvement of different transducers depending, at least in part, on the nature of the preconditioning stimulus. They include members of mitogenactivated protein kinases (MAPKs) and their phosphorylated Ras, Raf, MEK, and ERK subfamilies (7, 22, 24, 25); mitochondrial ATP-sensitive K+ (KATP) channels (26, 27) Akt (28–31); and the protein kinase C-*ε* isoform (32). The possibility that the nitric oxide (NO)-based adaptive response to hypoxia in *Drosophila* (33) is evolutionarily conserved suggests that this multifunctional modulator might be a logical choice as an autocrine and paracrine mediator of preconditioning stress. Indeed, pharmacological and genetic evidence supporting the involvement of NO in the transduction process is continuing to mount. As matter of fact, it has been reported that NO can exert a neuroprotective role during preconditioning (7, 34), and we have recently proposed that NO-induced neuroprotection is associated to preservation of mitochondrial function (24). On the other hand, preconditioning positively affects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia, an effect restricted to the delayed window of preconditioning (35). Moreover, preconditioning prevents mitochondrial swelling, preserves membrane integrity fluidity, and protects mitochondrial energy metabolism during cerebral ischemia avoiding ATP consumption (36).

Given the redox sensitivity of many of those above-mentioned kinases and transcription factors, reactive oxygen species might also be contemplated within transducers (37–39). Furthermore, adenosine, another prototypical paracrine mediator and "retaliatory metabolite," whose production is linked to ATP degradation, might be considered a transducer responsible for tolerance induction, in some *in vitro* and *in vivo* models (26, 27, 40, 41).

It is worth of note that although in general, mitochondria have been considered important mediators of endogenous neuroprotection, the mechanisms by which they might "integrate" cytoprotective signaling of preconditioning remain to be fully elucidated.

### **Role of mitochondria in ischemic tolerance**

It is well known that mitochondria are also able to sense and shape cytosolic Ca<sup>2</sup><sup>+</sup> signals by taking up and subsequently releasing Ca<sup>2</sup><sup>+</sup> ions during physiological and pathological Ca<sup>2</sup><sup>+</sup> elevations (42). Mitochondrial function is strictly dependent on maintaining of mitochondrial calcium homeostasis. In fact, thanks to the activity of Ca<sup>2</sup>+-sensitive mitochondrial dehydrogenases these organelles can regulate oxidative phosphorylation and ATP synthesis during conditions of high cellular demand (43). Therefore, to explore the relationship between mitochondrial oxidative capacity and calcium buffering activity during IPC might represent an attractive strategy mediating neuroprotection in ischemic conditions. In particular, considering that the influence of Ca<sup>2</sup><sup>+</sup> in the regulation of mitochondrial function is highly dependent on the spatiotemporal distribution of [Ca<sup>2</sup>+]<sup>i</sup> (44, 45), the assumption that during IPC an interaction between mitochondria and ER might affect mitochondrial metabolic properties, and in turn neuronal survival has been also investigated (31). This hypothesis is supported by the concept that microdomains of high [Ca<sup>2</sup>+]<sup>i</sup> have been identified near Ca<sup>2</sup><sup>+</sup> channels on the plasma membrane and ER (46).

# **Cellular Ionic Homeostasis and Energy Metabolism During Ischemic Preconditioning**

It has been widely described that IPC activates intracellular biological responses prior to a potential lethal insult (17, 47–49), and that these events make the tissue in which they occur more resistant to the subsequent severe ischemia (17, 47, 50–52). In this regard, it is possible to speculate that an increase of energy metabolism or a latency in anoxic depolarization following the onset of ischemic insult might represent one of the mechanisms by which tissues strengthen their tolerance when exposed to a sub-lethal insult. Indeed, it has been reported that a reduction in energy demand and in the activity of ion channels represents determinant factors for ischemic tolerance establishment (19, 53, 54). To this aim, *in vitro* experiments performed in cortical neurons demonstrated that the exposure to brief non-injurious oxygen and glucose deprivation (OGD) causes an impairment in voltage-gated potassium channels (55). Similarly, the impairment of Na+/K+- ATPase activity occurring in rat hippocampal and cortical neurons exposed to global forebrain ischemia is counteracted by IPC (56). Interestingly, in the last years, it has been shown that some integral plasma membrane proteins, involved in the control of Ca<sup>2</sup><sup>+</sup> and Na<sup>+</sup> ion influx or efflux, the sodium calcium exchangers (NCXs) might function as crucial players in the pathogenesis of brain ischemic damage (57). Therefore, these proteins, by regulating Na<sup>+</sup> and Ca<sup>2</sup><sup>+</sup> homeostasis, may represent more suitable molecular targets for therapeutic intervention in ischemic stroke. As matter of fact, *in vivo* experiments performed in gerbils demonstrated that during IPC, an increase in Ca<sup>2</sup>+-ATPase activity and an enhancement in mitochondrial calcium sequestration occur in CA1 hippocampal neurons (47). In line with this findings, intracellular calcium measurements in hippocampal neurons of preconditioned gerbils showed that the increase in [Ca<sup>2</sup>+]<sup>i</sup> occurring after anoxic and aglycemic episodes was markedly reduced in the ischemic tolerant animals (58). However, the molecular mechanisms responsible for this effect are still matter of debate and not completely clarified. As recently proposed by Kato et al.

(59), a possible explanation could be related to the increased expression of the Ca<sup>2</sup>+-ATPase isoform 1 [plasma membrane calcium ATPase 1 (PMCA-1)]. Moreover, considering that mitochondria play a crucial role in the regulation of intracellular Ca<sup>2</sup><sup>+</sup> homeostasis due to their high capacity to sequestrate Ca<sup>2</sup><sup>+</sup> both in physiological and in pathological conditions, they have been included among the possible intracellular mechanisms triggered by preconditioning stimuli. Indeed, although it is widely reported that the excessive amount of Ca<sup>2</sup>+, as it occurs during ischemia, causes an impairment of mitochondrial function with consequent massive uncoupling of oxidative phosphorylation, mitochondrial depolarization and mitochondrial permeability transitional pore (MPTP) opening, reversal of the action of ATP synthase and cell death, and studies performed both in the heart and in the brain demonstrated that the inhibition of MPTP opening and its signaling cascade represent crucial events responsible for cytoprotection observed in IPC (53, 60). What are the molecular mechanisms underlining these effects is still object of investigation. Among them, nitrite and protein kinases have been proposed as possible MPTP regulators (61–64) **Table 1**.

More interestingly, the hypothesis that a modulation of the expression and activity of the NCXs might have a role in the regulation of calcium and sodium homeostasis during ischemic tolerance has been recently suggested (31, 50, 71). This might be relating to the ability of Na+/Ca<sup>2</sup><sup>+</sup> exchanger to work in concert with selective ion channels and ATP-dependent pumps, in maintaining the physiological cytosolic concentrations of these two ions (72).

# **NCXs functional properties in ischemic brain**

In the brain, unlike other tissues, NCX is present in three different gene products, named NCX1, NCX2, and NCX3, with a

Sisalli et al. ER/mitochondria interaction in preconditioning-induced neuroprotection

distinct distribution pattern in different brain regions (73). Under physiological conditions, NCX works primary extruding Ca<sup>2</sup><sup>+</sup> in response to a depolarization or to an increase in intracellular Ca<sup>2</sup><sup>+</sup> concentrations coupled to receptor stimulation (74). However, during hypoxic conditions, when the dysfunction of the two plasma membrane ATP-dependent pumps Na+/K<sup>+</sup> ATPase and Ca<sup>2</sup><sup>+</sup> ATPase occurs, NCX assumes a relevant role in controlling the intracellular homeostasis of these two cations, since it is able to operate promoting Na<sup>+</sup> ions extrusion and Ca<sup>2</sup><sup>+</sup> influx (74, 75). Although this reverse mode of operation in the early phase of anoxia does undoubtedly elicit an increase in [Ca<sup>2</sup>+]<sup>i</sup> , its effect could be beneficial for neurons because it contributes to decrease [Na+]<sup>i</sup> overload, a phenomenon which would otherwise lead to cell swelling and thus to sudden necrotic neuronal death. Conversely, in the later phase of neuronal anoxia, when [Ca<sup>2</sup>+]<sup>i</sup> overload takes place, NCX working in the forward mode of operation can contribute to the lowering of Ca<sup>2</sup><sup>+</sup> concentrations, and thus it can protect neurons from intracellular Ca<sup>2</sup>+overload, neurotoxicity, and subsequent cell death (74). As we recently demonstrated, NCX1, NCX2, and NCX3 may exert different roles during *in vitro* and *in vivo* anoxic conditions leading to a new paradigm in the pathogenesis of ischemic damage. Indeed, we provided evidences that in cells singly and stably transfected with NCX3, this isoform contributes more significantly to the maintenance of [Ca<sup>2</sup>+]<sup>i</sup> homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial ΔΨ collapse and cell death (76). This is due to a different sensitivity of the three NCX isoforms to the changes in ATP content occurring during anoxia. Indeed, NCX3 results more resistant to ATP changes compared to NCX1 and NCX2 (76). In addition, in *in vivo* experiments, the selective knocking down of NCX1 and NCX3, but not of NCX2, by antisense oligodeoxynucleotide strategy (57) or the disruption of the *ncx3* gene, renders the brain more susceptible to the ischemic insult (77). Moreover, the induction of permanent middle cerebral artery occlusion in rats correlates with NCX1 mRNA upregulation in the peri-infarct area thus suggesting the possibility that this isoform could be a new druggable target for the treatment of cerebral ischemia. In line with this hypothesis, we recently demonstrated that NCX1 transcript and protein were upregulated by ischemic cerebral preconditioning. This effect was mediated by the transcriptional factor HIF1α and was accompanied by a relevant neuroprotection (71).

# **Role of NCX1 and NCX3 in the ischemic preconditioning**

Recent findings demonstrated that the two isoforms of the Na+/Ca<sup>2</sup><sup>+</sup> exchanger, NCX1 and NCX3, which are involved in several pathophysiological aspects of cerebral ischemia, can be included among the members of the growing family of the mediators involved in the ischemic brain tolerance due to their ability to regulate neuronal calcium homeostasis (**Figure 1**). In particular, we provide evidence that neuroprotection observed in preconditioned neurons exposed to OGD/reoxygenation is correlated to the increase in NCX1 and NCX3 protein expression. Indeed, the treatment with siRNA against NCX1 and NCX3 prevents this effect. These data are in accordance with results recently observed *in vivo* in an animal model of IPC (50). Consistently with these results, the upregulation of NCX1 and NCX3 protein expression in neurons exposed to IPC was dependent on PI3K/Akt activation, since the treatment with LY294002 was able to abolish this increase. Interestingly, we demonstrated that NO plays a key role in the triggering PI3K/Akt pathway as the increase in the phosphorylated form of Akt observed within 30 min after IPC was completely abolished by the treatment with L-NAME. More importantly, the treatment with L-NAME was able to inhibit NCX3 but did not affect NCX1 protein expression (31).

# **Calcium Homeostasis in ER and Mitochondria During Ischemic Preconditioning**

It has recently demonstrated that in neurons NCX3, apart its localization on the plasma membrane, is also expressed on the outer mitochondrial membrane where it contributes to the extrusion of calcium from mitochondria (78). It is well known that mitochondria, in addition to the generation of cellular energy, play an important role in regulating cellular calcium homeostasis (79–82) in concert with the sarco-endoplasmic reticulum Ca<sup>2</sup>+-ATPase (SERCA), the plasma membrane Ca<sup>2</sup>+-ATPase, and Na+/Ca<sup>2</sup><sup>+</sup> exchanger (83). On the other hand, the maintenance of mitochondrial calcium homeostasis is an important requirement ensuring mitochondrial function. In fact, Ca<sup>2</sup>+-sensitive dehydrogenases can regulate oxidative phosphorylation and ATP synthesis during times of high cellular demand (43). Therefore, it is possible to hypothesize that the increased expression of NCX3, we observed within 48 h from the sub-lethal insult, might exert neuroprotective effects regulating calcium handling and improving mitochondrial oxidative capacity. This finding is in line with the results previously obtained in an *in vitro* model of IPC and demonstrating that the increase in nNOS expression and NO*•* production through the activation of Ras/ERK1/2 pathway stimulated mitochondrial MnSOD. These effects associate with a reduction in free radical production and cytocrome *c* release from mitochondria to cytosol, and in turn with an improvement of neuronal survival (24). The demonstration that NCX3 might represent a target of IPC-induced neuroprotection (31) adds new insight into the molecular mechanisms involved in the ischemic brain preconditioning. Indeed, the over-expression of NCX3 might help mitochondria to preserve their energetic capacity making them less vulnerable to the subsequent lethal insult represented by OGD/reoxygenation. This hypothesis was strongly supported by the results obtained measuring the activity of NCX during IPC. In fact, we demonstrated that IPC induced an increase of NCX activity in the reverse mode of operation that was still observed in preconditioned neurons exposed to OGD/Reoxygenation. This is an early event since it occurred within 30 min after IPC stimulus was due to the contribution of NCX1 and NCX3 isoforms and was promoted by NO because it was abolished by the treatment with L-NAME. Intriguingly, this effect was associated with an increase in ER calcium content. Consistently with data previously published in our Laboratory (84), we speculated that NCX1, working in the reverse mode of operation, plays a key role in

the regulation of ER calcium refilling in the early phase of IPC (**Figure 2**). Indeed, the treatment with siNCX1, but not siNCX3, was able to prevent this phenomenon. The novel aspect of the study is the demonstration that NO promoted NCX1-induced ER refilling that was hampered by L-NAME pretreatment. This finding is in accordance with data recently published by Secondo et al. and demonstrating that NO was able to stimulate NCX1 to work in the reverse mode of operation, whereas NO did not affect NCX3 activity (85). The possibility that NCX1 activation in the early phase of IPC could affect mitochondrial calcium content promoting mitochondrial calcium uptake could not be excluded. However, in the late phase of IPC mitochondrial calcium handling is mainly regulated by NCX3 that is able to promote mitochondrial calcium extrusion (**Figure 2**). These data are supported by the

finding that NCX3 expression increased 48 h after the IPC insult (31). We have previously demonstrated that NCX3 is distributed also at mitochondrial level; therefore, it is possible to speculate that during IPC, the increased expression of NCX3 on mitochondria might contribute to the efflux of calcium from the organelle thus protecting neurons by the subsequent mitochondrial calcium overload induced by lethal OGD/reoxygenation exposure. The finding that the treatment with CGP and siNCX3 counteracted the effect of IPC on mitochondrial calcium content, leading to the lack of IPC-neuroprotection, further supports this hypothesis (31). Collectively, we can conclude that a functional interplay between NCX1 and NCX3 occurs during IPC. This phenomenon is tightly dependent on NO and Akt activation, and by contributing to the modulation of intracellular ionic homeostasis, could represent one

of the mechanisms responsible for neuroprotection induced by IPC (**Figure 2**).

# **Conclusion**

The experimental data described in this review support the hypothesis that mitochondria play a pivotal role in neuroprotective events provided by preconditioning. Indeed, both the mitochondrial antioxidant enzyme MnSOD and the proteins involved in the regulation of mitochondrial calcium homeostasis, such as mNCX3, represent new targets and mediators involved in preconditioning-induced neuroprotective responses. This is in line with the results reported in the literature that "mitochondrial preconditioning" has many neuroprotective effects, both during and following neurotoxic insults, including an improvement of neuronal viability, the attenuation of the intracellular Ca<sup>2</sup><sup>+</sup> influx, the suppression of ROS generation, the inhibition of apoptosis, and the maintenance of ATP levels (86). Therefore, understanding the precise mitochondrial mechanisms involved in preconditioning will provide important information necessary to develop new and more effective therapeutic strategies for neurodegeneration occurring in brain ischemia. In this regards, the identification of NCX3 as new player in the regulation of mitochondrial calcium efflux and the demonstration that an increase in its expression occurs during preconditioning, promoting mitochondrial calcium homeostasis, might help to draw new therapeutic strategy potentially able to delay neuronal loss occurring in ischemia. More interestingly, the demonstration that an increase in NCX1 and NCX3 activity is responsible for Ca<sup>2</sup><sup>+</sup> cycling from ER and mitochondria, and proof that it activates intracellular events leading to neuroprotection observed in IPC support this hypothesis (31). In this scenario, the identification of a compound able to selectively stimulate the activity of NCX1 and to prevent neuronal degeneration in *in vitro* and *in vivo* models of ischemia has been recently synthesized (77). Moreover, compounds able to stimulate NCX3 activity are in progress of development in our Laboratory.

Therefore, based on this early observation, these studies have potentially revealed new molecular targets in cerebral ischemia and neurodegenerative diseases pathogenesis, which ultimately may open up alternative avenues for future therapeutic intervention.

# **Acknowledgments**

This work was supported by grants from Regione Campania #PON01\_01602 and PON03\_00785 to Lucio Annunziato.

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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 Sisalli, Annunziato and Scorziello. 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.*