low ambient GABA represents an inadequate situation to activate GABAA receptors mediating tonic inhibition with a consequent facilitation of seizure activity, depression-like, and anxiety-like behaviors (Merali et al., 2004; Maguire and Mody, 2008; Hines et al., 2012). Our experiments revealed reduced GABA tissue levels in the neocortex of patients with TLE and comorbid anxiety and depression, a situation that can be associated with the low GABA release in pharmacoresistant epilepsy described by other authors (During and Spencer, 1993; Luna-Munguia et al., 2011).

Activation of GABAB receptors starts several signaling cascades at pre- and postsynaptic levels essential for the stability of the cortical network activity and modulation of gamma oscillations essential for cognitive processes (Kohl and Paulsen, 2010). In contrast, a deficiency in the neurotransmission mediated by GABAB receptors has been associated with changes in cortical gamma oscillations with consequent psychiatric disorders such as schizophrenia (Uhlhaas and Singer, 2010). We found that patients with TLE without anxiety and/or depression presented decreased GABAB-induced G-protein activation restricted to layers V–VI. In contrast, patients with TLE and comorbid anxiety and/or depression showed reduced GABAB receptor-induced G-protein activation in all cortical layers. These findings associated with low GABA tissue content suggest a deficit in the neurotransmission induced by GABAB receptors in temporal cortex of patients with TLE and comorbid anxiety and/or depression, a situation similar to that found in patients with mood disorders (Enna and Bowery, 2004; Croarkin et al., 2011). Indeed, the high GABAB binding found in cortical layers III–IV and V–VI (present study), as well as the up-regulation of GABAB in remaining neurons in hippocampus (Princivalle et al., 2002) can represent a compensatory mechanism resulting from the deficient neurotransmission mediated by these receptors in patients with TLE.

The comorbidity of mood disorders in patients with epilepsy has been associated with a higher perception of side effects of the therapy with AEDs (Gómez-Arias et al., 2012). AEDs therapy can induce uncoupling of GABA/BZD site interactions and alterations in GABAA and GABAB function (Mula and Sander, 2007); these changes may result from the repetitive administration of BDZs and/or AEDs that enhance GABA exposure such as tiagabine and vigabatrin (Suzuki et al., 1991; Gravielle et al., 2005; Perici´c et al., 2007). Carbamazepine and valproic acid increase the number of GABAB binding sites in the rat hippocampus when applied chronically, an effect that has been associated with the mood stabilizing effects induced by these drugs (Motohashi,1992). In contrast, long-term exposure to these AEDs may decrease the GABAB receptor function, an effect analogous to that produced by the subchronic administration of baclofen (Pacey et al., 2011). Our results do not support a significant association between the pharmacotherapy with AEDs inducing GABAergic effects in temporal neocortex and the comorbid anxiety and/or depression in patients with TLE. However, future studies including other brain areas and a larger number of patients should be carried out to support this hypothesis.

Affective disorders have been associated with disturbances in the second messenger signaling via G-protein function (Pacheco et al., 1996). Hyperfunction of G proteins leads to characteristics of a manic or depressive state caused by instability in the activities of protein kinases C (Avissar and Schreiber, 1992), a family of enzymes that are involved in the signal transduction mechanism of the GABAB receptors (Taniyama et al., 1992). This study indicates an imbalance of the G-protein activation induced by stimulation of GABAB receptors in the temporal neocortex of patients with TLE and comorbid anxiety and/or depression. This situation should be kept in mind when considering these receptors as promising targets for the therapy of psychiatric disorders associated with TLE.

The comorbidity of mood disorders in TLE has also been related to a higher seizure frequency (Grabowska-Grzyb et al., 2006; Mula and Sander, 2007; Peng et al., 2014) and longer duration of active epilepsy (Gonçalves and Cendes, 2011). Our results revealed that the lower values for BDZ binding, GABABinduced G-protein activation, and GABA tissue content correlated with a higher seizure frequency and the longer duration of epilepsy of patients with TLE and comorbid anxiety and/or depression. In contrast, the higher BDZ binding and elevated GABAB-induced G-protein activation correlated with a lower seizure frequency of patients with TLE without psychiatric disturbances. It is possible that the higher seizure frequency and longer duration of epilepsy augment the exposure to elevated extracellular GABA levels during the ictal period. This situation may result in GABAA desensitization, dysregulation of the neurotransmission mediated by GABAB receptors, and uncoupling of GABA/BZD site interactions (Gravielle et al., 2005), conditions that could facilitate the comorbid anxiety and/or depression of patients with TLE. Finally, further experiments should be carried out to identify if the receptor binding alterations detected in the present study are the consequence of changes in the number or affinity of the receptors evaluated. We also suggest future studies in patients with anxiety and depression disorders but without epilepsy to explain the findings obtained in the present study.

#### **AUTHOR CONTRIBUTIONS**

The author Luisa Rocha designed the study and prepared the manuscript. The author Mario Alonso-Vanegas performed the epilepsy surgery to all the patients. The author Iris E. Martínez-Juárez carried out the pre-surgical evaluation of the patients with pharmacoresistant epilepsy. The authors Sandra Orozco-Suárez, David Escalante-Santiago, and Iris Angélica Feria-Romero performed the semiquantitative RT-PCR analysis. The author Cecilia Zavala-Tecuapetla carried out the functional autoradiography experiments. Under the supervision of Mario Alonso-Vanegas, the authors José Miguel Cisneros-Franco, Ricardo Masao Buentello-García, and Jesús Cienfuegos analyzed the clinical data of the patients and their correlation with the results obtained. All authors contributed to manuscript revisions and have approved the final manuscript.

#### **ACKNOWLEDGMENTS**

We thank Francia Carmona and the staff members of the outpatient Epilepsy Surgery Clinic for their support. This study received funding from Consejo Nacional de Ciencia y Tecnología (Grant 98386).

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fncel.2014.00442/ abstract

### **REFERENCES**

**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.

*Received: 18 October 2014; accepted: 08 December 2014; published online: 05 January 2015.*

*Citation: Rocha L, Alonso-Vanegas M, Martínez-Juárez IE, Orozco-Suárez S, Escalante-Santiago D, Feria-Romero IA, Zavala-Tecuapetla C, Cisneros-Franco JM, Buentello-García RM and Cienfuegos J (2015) GABAergic alterations in neocortex of patients with pharmacoresistant temporal lobe epilepsy can explain the comorbidity of anxiety and depression: the potential impact of clinical factors. Front. Cell. Neurosci. 8:442. doi: 10.3389/fncel.2014.00442*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2015 Rocha, Alonso-Vanegas, Martínez-Juárez, Orozco-Suárez, Escalante-Santiago, Feria-Romero, Zavala-Tecuapetla, Cisneros-Franco, Buentello-García and Cienfuegos. 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.*

# HDAC4 as a potential therapeutic target in neurodegenerative diseases: a summary of recent achievements

#### *Michal Mielcarek1 \*, Daniel Zielonka2, Alisia Carnemolla1, Jerzy T. Marcinkowski 2 and Fabien Guidez 3*

*1 Department of Medical and Molecular Genetics, King's College London, London, UK*

*2 Department of Social Medicine, Poznan University of Medical Sciences, Poznan, Poland*

*3 INSERM UMRS 1131, Université Paris Diderot, Institut Universitaire d'hématologie (IUH), Hôpital Saint-Louis, Paris, France*

#### *Edited by:*

*Marco Antonio Meraz-Ríos, Centro De Investigación Y De Estudios Avanzados, Mexico*

#### *Reviewed by:*

*Vincenzo De Paola, Imperial College London, UK Henry Markram, Ecole Polytechnique Federale de Lausanne, Switzerland*

#### *\*Correspondence:*

*Michal Mielcarek, Department of Medical and Molecular Genetics, School of Medicine, King's College London, 8th Floor Tower Wing, Guy's Hospital Great Maze Pond, London, SE1 9RT, UK e-mail<EMAIL_ADDRESS>
For the past decade protein acetylation has been shown to be a crucial post-transcriptional modification involved in the regulation of protein functions. Histone acetyltransferases (HATs) mediate acetylation of histones which results in the nucleosomal relaxation associated with gene expression. The reverse reaction, histone deacetylation, is mediated by histone deacetylases (HDACs) leading to chromatin condensation followed by transcriptional repression. HDACs are divided into distinct classes: I, IIa, IIb, III, and IV, on the basis of size and sequence homology, as well as formation of distinct repressor complexes. Implications of HDACs in many diseases, such as cancer, heart failure, and neurodegeneration, have identified these molecules as unique and attractive therapeutic targets. The emergence of HDAC4 among the members of class IIa family as a major player in synaptic plasticity raises important questions about its functions in the brain. The characterization of HDAC4 specific substrates and molecular partners in the brain will not only provide a better understanding of HDAC4 biological functions but also might help to develop new therapeutic strategies to target numerous malignancies. In this review we highlight and summarize recent achievements in understanding the biological role of HDAC4 in neurodegenerative processes.

**Keywords: histone deacetylase, signaling, HDAC4, neurodegeneration, HDAC inhibitors, therapeutic potential**

#### **INTRODUCTION**

Transcription is a multistep process and its regulation involves a balanced coordination of several molecular factors. Epigenetic modifications of chromatin, including histone acetylation, represent priming events in the cascade leading to gene expression and are governed by the antagonistic activity of two families of enzymes: the histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Fischer et al., 2010). The covalent modification of conserved lysine residues within histone proteins by acetyl groups leads to a nucleosomal relaxation and transcriptional activation; this reversible process provides a central mechanism to control gene expression and cellular signaling events. As such HDACs mediate epigenetic mechanisms that play a key role in homeostasis of histone functions and gene transcription. Mammalian HDACs are a family of 18 proteins divided into four groups based on structural and functional similarities: class I (HDACs: 1, 2, 3, 8), class IIa (HDACs: 4, 5, 7, 9), class IIb (HDACs: 6, 10), class III (sirtuins 1-7), and HDAC11 as the sole member of class IV (Saha and Pahan, 2006). It is well established that HDACs alter cell growth and differentiation by either governing chromatin structure or repressing the activity of specific transcription factors (Fischer et al., 2010). They are often deregulated in diseases and inhibition of their enzymatic activities remains of therapeutic interest.

Interestingly, the class IIa subgroup of HDACs shows a number of unique features in comparison to other HDACs (Verdin et al., 2003). Unlike the class I enzymes that are predominantly localized in the nucleus, class IIa HDACs shuttle between the nucleus and cytoplasm, a process that is controlled through the phosphorylation of specific serine residues (Chawla et al., 2003). The class IIa members are potent transcriptional repressors due to a high interaction propensity of N-terminal domains toward tissue specific transcription factors (Petrie et al., 2003; Saha and Pahan, 2006). Finally, the C-terminal catalytic domain of class IIa enzymes is characterized by a unique mutation of Tyr residue into His leading to the deacetylase domain inactivation (Lahm et al., 2007), emphasizing their distinctive properties compared to other HDACs (Verdin et al., 2003; Saha and Pahan, 2006).

HDAC4 shows approximately 60–70% sequence identity to HDAC5 and HDAC7 (de Ruijter et al., 2003). Among class IIa HDACs, HDAC4 has been described as a potent transcriptional repressor that is able to interact via its N-terminal domain with many different co-repressors, specifically in the brain. Hence, HDAC inhibitors have been recently used in the treatment of a wide-range of brain disorders characterized by pathological alterations in the transcriptome (Fischer et al., 2010) and they have displayed neuroprotective effects in animal models of such neurological disorders like Huntington's disease (HD), Alzheimer's disease (AD) and ischemic stroke.

#### **REGULATION OF HDAC4 EXPRESSION, CELLULAR LOCALIZATION AND FUNCTION**

HDAC4 is ubiquitously expressed throughout the body with enrichment in the brain, heart and skeletal muscle (Verdin et al., 2003; Broide et al., 2007). However, the transcriptional mechanisms involved in the regulation of HDAC4 expression are poorly understood. Recent studies have shown that HDAC4 expression might be tightly regulated by microRNAs. In podocytes, miR-29a reduced HDAC4 signaling and attenuated the glucocorticoidmediated β-catenin deacetylation and ubiquitination (Ko et al., 2013; Lin et al., 2014). Similarly, miR-365 reduced endogenous HDAC4 protein levels and led to inhibition of chondrocyte differentiation (Guan et al., 2011). Overexpression or inhibition of miR-206 in skeletal muscles has been associated with a decrease or increase of endogenous HDAC4 levels, respectively (Williams et al., 2009; Liu et al., 2012; Winbanks et al., 2013). HDAC4 expression has also been reported to be up-regulated under ER stress through its interaction with activating transcription factor 4 (ATF4). *In vitro*, HDAC4 overexpression caused ATF4 cytoplasmic retention and inhibition of ATF4 transcriptional activity, suggesting the presence of an autoregulatory loop. ER stress can ultimately promote cell apoptosis through up-regulation of ATF4 target genes such as CHOP and TRB3 and this effect was exacerbated by HDAC4 down-regulation (Zhang et al., 2014).

It is believed that HDAC4 undergoes a signal-dependent shuttling between the cytoplasm and nucleus, although in the brain it seems to be exclusively localized to the cytoplasm (Darcy et al., 2010; Mielcarek et al., 2013a,b). This nuclear-cytoplasmic shuttle is controlled by multiple mechanisms including activity of calcium/calmodulin-dependent kinase (CaMK) (Bolger and Yao, 2005) and salt-inducible kinases (Walkinshaw et al., 2013a), cAMP signaling (Walkinshaw et al., 2013b), and oxidative stress (Matsushima et al., 2013) (reviewed in Parra and Verdin, 2010, **Figure 1A**). Under normal conditions phosphorylated HDAC4 was retained in the cytoplasm through its association with 14-3-3 proteins (Grozinger and Schreiber, 2000; Verdin et al., 2003), (**Figure 1A**) and once dephosphorylated at Ser298 by the catalytic subunit of PP2A (Protein Phosphatase 2) it moved into the nucleus (Paroni et al., 2008).

It has been shown that HDAC4 can produce three specific nuclear pools including full length HDAC4 and two N-terminal fragments with different functions controlling cell death and differentiation *in vitro* (Paroni et al., 2004, 2007; Backs et al., 2011). Indeed, HDAC4 protein can be cleaved by caspases leading to a HDAC4-nuclear fragment generation (Paroni et al., 2004, 2007). Cleavage of HDAC4 occurred at Asp289 and resulted in the formation of a cytosolic carboxy-terminal fragment and an amino-terminal fragment that accumulated into the nucleus. This nuclear fragment exhibited a stronger cell death-promoting activity coupled with increased repressive effect on Runx2 (Runtrelated transcription factor 2) or SRF (Serum response factor) dependent transcription. Interestingly, this nuclear fragment was a less potent inhibitor of MEF2C (Myocyte enhance factor 2C) driven transcription, compared to full-length HDAC4 (Paroni et al., 2004), although such repressor activity has been described as independent from the acetylase domain. While caspase-2 and caspase-3 have been shown to cleave HDAC4 *in vitro,* caspase-3 was critical for HDAC4 cleavage *in vivo* during UV-induced apoptosis (Paroni et al., 2004). In the nucleus, a caspase-generated HDAC4 fragment was also reported to trigger cytochrome C release from mitochondria and cell death in a caspase-9 dependent manner (Liu and Schneider, 2013). In isolated skeletal muscle fibers expressing a HDAC4-green fluorescent protein, activation of PKA by the beta-receptor agonist isoproterenol or dibutyryl cAMP caused a steady HDAC4 nuclear influx. Thus, mutations of Ser265 and Ser266 (PKA targeted serines) enabled HDAC4 to respond to PKA activation (Liu and Schneider, 2013). Similarly, clenbuterol a potent β2-adrenoreceptor stimulator in skeletal muscles caused HDAC4 phosphorylation on Ser246 through activation of CaMKII (Ohnuki et al., 2014). In cardiomyocytes, PKA induced generation of the N-terminal HDAC4 cleavage product at Tyr202. This N-terminal fragment selectively inhibits activity of MEF2 but not SRF, thereby antagonizing a pro-hypertrophic potential of CaMKII signaling without affecting cardiomyocytes survival. Thus, HDAC4 may function as a molecular nexus for the antagonistic actions of the CaMKII and PKA pathways (Backs et al., 2011). In addition, sustained glycolysis induced by lipopolysaccharide (LPS) treatment activated caspase-3, which cleaved HDAC4 and triggered its degradation. Importantly, a caspase-3 resistant HDAC4 mutant escaped LPS-induced degradation and prolonged inflammatory cytokine production through the GSK3β (Glycogen Synthase Kinase-3 β isoform)–iNOS (inducible Nitric Oxide Synthase)–NO (Nitric Oxide) axis (Wang et al., 2014a). However, until now, there have been no data available suggesting a similar proteolytic pattern of HDAC4 in the healthy brain or in neurodegenerative disorders.

Interestingly, cleavage and phosphorylation sites are all located within the N-terminal region of HDAC4 protein highlighting this area as an important regulatory domain. While this N-terminal region seems to be critical for the repressive function of HDAC4, it also contains a transcription factor interacting domain that can bind MEF2 family members. HDAC4-MEF2 interaction was associated with the inhibition of MEF2 function resulting in neuronal cell death (Mao et al., 1999) and repression of MEF2-dependent genes in neuronal cells (Bolger and Yao, 2005) and skeletal muscles (Miska et al., 2001). In addition, the HDAC4 N-terminal region is characterized by a high glutamine content that is likely responsible for interactions with other glutamine-rich proteins leading to a spontaneous assembly of insoluble toxic amyloid-like structures (Fiumara et al., 2010). X-ray resolution of the human HDAC4 glutamine-rich domain showed that this domain is preferentially folding into a straight alpha-helix which assembles into a tetramer. In contrast to the coiled coil proteins, the HDAC4 tetramer lacked the regular arrangement of apolar residues and had an extended hydrophobic core that might lead to its rapid equilibrium with monomer and intermediate species (Guo et al., 2007). Overall, these studies provide a picture of a multifunctional protein and emphasize the presence of several mechanisms behind the tissue-specific regulation of HDAC4 expression and function.

# **REGULATION OF HDAC4 DEACETYLASE ACTIVITY**

Previous findings have suggested that class IIa HDACs are inactive on acetylated substrates, thus differing from class I and IIb enzymes (de Ruijter et al., 2003). HDAC4 catalytic domain purified from bacteria was 1000-fold less active than class I HDACs on standard substrates (Lahm et al., 2007). In contrast to the other HDACs, the C-terminal catalytic domain of the class IIa enzymes contains an amino acid substitution of a critical Tyr residue into His (Lahm et al., 2007). Mutation of this Tyr to His in class I HDACs severely reduced their activity, while a His-976- Tyr mutation in HDAC4 produced an enzyme with a 1000-fold higher catalytic efficiency (Lahm et al., 2007). Interestingly, mutations in the residues involved either in the coordination of the structural zinc binding domain of HDAC4 or the binding site of class IIa selective inhibitors prevented the association of HDAC4 with N-CoR/HDAC3 associated repressor complex (Bottomley et al., 2008). It has been proposed that HDAC4 binds directly to HDAC3 in order to activate its deacetylase domain (Mihaylova et al., 2011) and that the structural zinc-binding domain is crucial in the regulation of class IIa HDAC functions (Bottomley et al., 2008).

Finally, HDAC4 activity seems to be modulated by the ubiquitin-proteasome system. Serum starvation elicited the polyubiquitination and degradation of HDAC4 in nontransformed cells. Phosphorylation of Ser298 within the PEST1 sequence, a GSK3β consensus sequence, played an important role in the control of HDAC4 stability. Phosphorylation of HDAC4 by GSK3β has been described to occur *in vitro* upon phosphorylation of Ser302, which seems to play a role of a priming phosphate (Cernotta et al., 2011) and removal of growth factors fails to trigger HDAC4 degradation in cells deficient in this kinase (**Figure 1B**). One might conclude that HDAC4 is not a histone/protein deacetylase, however it can play a crucial role in many processes through its interaction with HDAC3 or with a general role of scaffolding protein.

# **HDAC4 BIOLOGICAL FUNCTION IN NON CNS ORGANS**

As mentioned, HDAC4 is ubiquitously expressed, however, its initial biological function was described in chondrocytes: direct inhibition of RUNX2 by HDAC4 led to chondrocyte hypertrophy (Vega et al., 2004). HDAC4-null mice displayed premature ossification of developing bones due to an ectopic and early onset chondrocyte hypertrophy, mimicking the phenotype associated with the constitutive *Runx2* expression in chondrocytes. On the other hand, overexpression of HDAC4 in proliferating chondrocytes *in vivo* inhibited their hypertrophy and differentiation, mimicking a *Runx2* loss-of-function phenotype (Vega et al., 2004).

HDAC4 has been described as a critical factor that connects neural activity to the muscle remodeling program, nevertheless, its role in the physiology of this peripheral tissue is controversial and has not been entirely clarified. Inactivation of HDAC4 suppressed denervation-induced muscle atrophy while increased re-innervation (Williams et al., 2009; Winbanks et al., 2013). Although it has been observed that miR-206 could regulate HDAC4 expression in skeletal muscles, the postnatal expression of miR-206 is not a key regulator of a basal skeletal muscles mass or specific pathways of muscle growth and wasting (Winbanks et al., 2013). Previous studies established that muscle denervation strongly induced the expression of Gadd45a,

a small myonuclear protein that is required for denervationinduced muscle fiber atrophy. Interestingly, it was shown that HDAC4 mediated an induction of Gadd45a mRNA in denervated skeletal muscles (Bongers et al., 2013). Furthermore, HDAC4 has been described to induce AP1-dependent transcription by activating the HDAC4-MAPK-AP1 signaling axis essential for the neurogenic muscles atrophy (Choi et al., 2012). Interestingly, AP1 inactivation recapitulates HDAC4 deficiency and blunts the muscle's atrophy program. Surprisingly, HDAC4 stimulated AP1 activity by activating the HDAC4-MAPK-AP1 signaling axis essential for the neurogenic skeletal muscles atrophy (Choi et al., 2012). HDAC4 was also described as a member of the MEF2C repressor complex together with HDAC3 and Ser/Thr kinase homeodomain-interacting protein kinase 2 (HIPK2) in undifferentiated myoblasts (de la Vega et al., 2013). On the other hand, a recent study has shown that HDAC4 inactivation led to defective satellite cells proliferation, muscle regeneration and lipid accumulation (Choi et al., 2014).

Finally, it was shown that HDAC4 up-regulation was significantly greater in patients with rapidly progressive ALS (Amyotrophic lateral sclerosis) and its expression was negatively correlated with a degree of skeletal muscles re-innervation and functional outcome (Bruneteau et al., 2013). Similarly an increased level of HDAC4 has been found in SMA (Spinal muscular atrophy) model mice and in SMA patient muscles (Bricceno et al., 2012). Moreover, HDAC4 expression was increased in masseter muscles from a patient with a deepbite and was found to correlate negatively with slow myosin type I and positively with fast myosin type IIX (Huh et al., 2013). Overall, these studies provide evidence of an active role of HDAC4 in the neurogenic muscle's atrophy program, which becomes exacerbated in some neurodegenerative disorders, therefore, urging HDAC4 as a promising therapeutic target.

#### **HDAC4 FUNCTION IN THE BRAIN AND ITS IMPLICATION IN NEURODEGENERATION**

Compared to the other class IIa enzymes, HDAC4 is highly expressed in the mouse brain (Grozinger et al., 1999; Darcy et al., 2010) with a highest expression occurring during early postnatal life (Sando et al., 2012). Immunohistochemical analysis of brain sections revealed accumulation of HDAC4 in the cytoplasm of neurons, including neurons containing CRH, oxytocin, vasopressin, orexin, histamine, serotonin, and noradrenaline (Takase et al., 2013). HDAC4-immunoreactive puncta were uniform in size and were widely distributed in the neuropil of brain areas, including the PVN (Hypothalamic Paraventricular Nucleus), LHA (Lateral Hypothalamic Area), ARC (Hypothalamic Arcuate Nucleus), TMN (Tuberomammillary Nucleus), DR (Dorsal Raphe), and LC (Locus Coeruleus). Interestingly, these HDAC4 positive accumulations co-localized with PSD95-immunoreactive puncta (Mielcarek et al., 2013a; Takase et al., 2013 ), suggesting a role for HDAC4 in synaptic plasticity (Sando et al., 2012; Mielcarek et al., 2013a).

In neurons, dynamic changes in the subcellular localization of HDACs are thought to contribute to various signaling pathways. Treatment with the neuronal survival factor BDNF (Brain-derived neurotrophic factor) suppressed HDAC4 nuclear translocation, whereas a pro-apoptotic CaMK inhibitor stimulated HDAC4 nuclear accumulation. Moreover, as expected, an ectopic expression of the nuclear-localized HDAC4 led to neuronal apoptosis and repressed the transcriptional activities of survival factors in neurons like: MEF2 and cAMP response element-binding protein (CREB). In contrast, inactivation of HDAC4 by small interfering RNA or HDAC inhibitors suppressed neuronal cell death (Bolger and Yao, 2005). In cultured hippocampal neurons, localization of HDAC4 has been shown to be very dynamic and signal-regulated and spontaneous electrical activity was sufficient for HDAC4 nuclear export (Chawla et al., 2003). On the other hand, in various experimental models, it has been shown that loss of HDAC4 could lead to neurodegeneration of the retina (Chen and Cepko, 2009) and cerebellum (Majdzadeh et al., 2008). This might be explained by the chondrocyte hypertrophy that occurred in mice lacking HDAC4 causing developmental brain abnormalities due to a skull deformation (Vega et al., 2004) and might be further supported by major pathological changes in the *Hdac4* knock-out murine postnatal brain (Majdzadeh et al., 2008). In addition, a conditional knockout of *Hdac4* under the *CamkII* promoter in the mouse forebrain, showed impairments in the hippocampal-dependent learning and memory with a simultaneous increase in locomotor activity (Kim et al., 2012). However, it was recently shown that a selective deletion of *Hdac4* under the control of the *Thy1* or Nestin promoters resulted in a normal gross brain morphology and cytoarchitecture as well in a normal locomotor activity (Price et al., 2013). Moreover, the Affymetrix array data showed no effect of *Hdac4* knock-out on the transcriptional profile and global acetylation of the postnatal murine brain (Mielcarek et al., 2013b). Similarly, a hippocampal depletion of HDAC4 *in vivo* abolished long-lasting stress-inducible behavioral changes and improved stress related learning and memory impairments in mice (Sailaja et al., 2012). HDAC4 overexpression has been shown to accelerate the death of cerebellar granule neurons (Bolger and Yao, 2005; Li et al., 2012; Sando et al., 2012) by increasing their vulnerability to H202 insult due to an inhibition of PPARα activity (peroxisome proliferatorsactivated receptor α) (Yang et al., 2011). In addition, the HDAC4 viral-mediated overexpression in the rat hippocampus was sufficient to induce depression like behavior (Sarkar et al., 2014). Interestingly, overexpression of HDAC4 in the adult mushroom body, an important structure for memory formation, resulted in a specific impairment in long-term courtship memory but had no affect on short-term memory in *Drosophila* model (Fitzsimons et al., 2013). Similarly, HDAC4 and HDAC5 increased a cell viability through an inhibition of HMGB1, a central mediator of tissue damage following acute injury and it has been shown that NADPH oxidase-mediated HDAC4 and HDAC5 expression contributed to the cerebral ischemia injury through the HMGB1 signaling pathway that could be an effective therapeutic target to treat stroke (He et al., 2013).

# **HDAC INHIBITORS AND INHIBITION OF HDAC4 ACTIVITY**

Suberoylanilide hydroxamic acid, known also as SAHA or vorinostat, was the first HDAC inhibitor (HDACI) to be approved for the cancer therapy of advanced cutaneous T-cell lymphoma (Marks and Breslow, 2007). Initially, SAHA was identified as an inhibitor of class I and class II HDACs at nanomolar concentrations (Richon et al., 1998), but was further characterized as inhibitor of class I HDACs as well specifically HDAC6 within the class IIb enzyme (Parmigiani et al., 2008; Marks and Xu, 2009). More recently activity based probes have been used to demonstrate that SAHA can bind directly to both class I and IIa HDACs (Salisbury and Cravatt, 2007; Codd et al., 2009). Experiments performed on cancer cell lines revealed the ability of SAHA to induce the degradation via RANBP2-mediated proteasome of both HDAC4 and HDAC5 *in vitro* (Scognamiglio et al., 2008). Interestingly, other HDACIs, such as trichostatin A (TSA) and sodium butyrate, have also been reported to induce a reduction in HDAC4 levels when administered to embryoid bodies (Chen et al., 2011), suggesting that similarly to SAHA, these other HDACIs could induce HDAC4 degradation through a proteasome-dependent mechanism.

An increased expression of HDAC4 has been described in several *in* vitro and *in* vivo models of neuro-like disorders. Treatment of neuronal cell lines with SAHA led to a noticeable improvement of cell polarity and morphology, with longer processes in the rat H19-7 hippocampal cell line with folate deficiency. In this neuronal cell model, folate deficit led to a reduction in cell proliferation and decreased production of S-adenosylmethionine (the universal substrate for transmethylation reactions) concurrent with an increased expression of HDACs (including HDAC4,6,7) (Akchiche et al., 2012). Furthermore, cecal ligation and peroration (CLP) rats, used in the Sepsis-associated encephalopathy (SAE) study, were also characterized by an increased expression of HDAC4 (Fang et al., 2014). Administration of HDACIs (e.g., TSA or SAHA) restored Bcl-XL and Bax levels *in vivo* and decreased apoptotic cells *in vitro*. In addition, knock-down of HDAC4 by shRNA resulted in an enhanced histone acetylation like: H3 and H4 and reduced neuronal apoptosis. Consistently, CLP rats treated with TSA or SAHA exhibited significant spatial learning and memory deficits with no effect on their locomotive activity (Fang et al., 2014).

In preclinical settings, SAHA and other HDACIs have consistently improved the phenotype in HD mouse models (Ferrante et al., 2003; Hockly et al., 2003; Gardian et al., 2005) and are being developed as HD therapeutics. Recent findings have increasingly described a widespread peripheral organ pathology in HD, such as skeletal muscles atrophy (Zielonka et al., 2014a) and heart failure (Mielcarek et al., 2014a; Zielonka et al., 2014b), often associated with an increased HDAC4 expression (Mielcarek et al., 2014b). As such, class IIa HDACs inhibitors might be beneficial in delaying HD-related symptoms and, therefore, are under evaluation as HD therapeutics. It has been shown that the administration of SAHA to wild type and R6/2 mice decreased HDAC2 and HDAC4 at the protein but not RNA levels in different brain regions *in vivo* (Mielcarek et al., 2011), supporting previous observations from cancer cell lines (Scognamiglio et al., 2008). We have also shown that HDAC4 associates with huntingtin in a polyglutamine-length dependent manner and co-localizes with cytoplasmic inclusions. Consequently, reduction of HDAC4 levels delayed cytoplasmic aggregate formation in different brain regions of R6/2 mice and rescued cortico-striatal neuronal synaptic function in HD mouse models. This was accompanied by an improvement in motor co-ordination, neurological phenotypes and increased lifespan (Mielcarek et al., 2013a). Interestingly, SAHA treatment of R6/2 mice was accompanied by restoration of brain-derived neurotrophic factor (BDNF) cortical transcript levels (Mielcarek et al., 2011). An increased expression of BDNF has been associated with memory-enhancing and neuroprotective properties of HDACIs, as it has been shown that HDAC4 and HDAC5 might repress specific *Bdnf* transcripts in rats and primary hippocampal neuronal cultures and this effect was reversed by SAHA treatment (Koppel and Timmusk, 2013). However, the mechanism of BDNF induction by HDACIs is not yet fully understood. Surprisingly, HDAC4 reduction had no effect on global transcriptional dysfunction and did not modulate nuclear huntingtin aggregation in HD mousse models (Mielcarek et al., 2013a). Interestingly, elevated HDAC4 levels have been shown in *post mortem* HD (Yeh et al., 2013) and FTLD (Frontotemporal Lobar Degeneration) (Whitehouse et al., 2014) brains and HDAC4 has been described as a component of Lewy Bodies in Parkinson's disease brains (Takahashi-Fujigasaki and Fujigasaki, 2006) and of intranuclear inclusions in the neuronal intranuclear inclusion disease (Takahashi-Fujigasaki et al., 2006). Consistently, administration of SAHA has been shown to improve synaptic plasticity and learning behavior in an Alzheimer disease model (Kilgore et al., 2010). A causative role for HDAC4 has been also described in SCA-1 (Spinocerebellar Ataxia Type 1) as a modulator of Ataxin-1. It was shown that ataxin-1 bound specifically to HDAC4 and MEF2 and co-localized with them in the nuclear inclusion bodies. Significantly, these interactions were greatly reduced by the S776A mutation, which largely abrogates the cytotoxicity of ataxin-1 *in vitro* (Bolger et al., 2007). Moreover, HDAC4 has been found to be significantly overexpressed in specific cortical regions of autistic patients (Nardone et al., 2014).

Diabetes is one of major risk factors for dementia. However, the molecular mechanism underlying the risk of diabetes for dementia is largely unknown. Surprisingly, it has been shown that diabetes may cause epigenetic changes in the brain, which adversely affect synaptic function. These alterations were associated with an increased susceptibility to oligomeric Aβ-induced synaptic impairments in the hippocampal structure that eventually led to synaptic dysfunction. Use of pharmacological inhibitors against the HDAC IIa family restored synaptic function (Wang et al., 2014b). This therapeutic effect highlights the importance of HDACIIa members, including HDAC4, as a possible target in the brain.

# **CONCLUSION**

There is convincing evidence suggesting that HDAC4 plays a central role in the brain physiology and that it is deregulated in several neurodegenerative disorders, therefore representing a suitable therapeutic target, through which HDACs inhibition may occur. However, the use of currently available HDACIs is likely to involve adverse side effects due to the broad spectrum HDAC inhibition. Therefore, a search for selective HDAC inhibitors would likely be of benefit for targeted therapy. Likely, HDACs inhibition occurs through a dual mechanism, either by a direct inhibition of an active deacetylase domain (class I, IIb, III, and IV) or by a direct binding followed by proteosomal degradation (class IIa) (**Figure 2**).  Although, class I HDACs biological functions have been intensively studied in the brain, it appears that suitable HDAC targets could arise from HDAC IIa subfamily but their function and role are still poorly understood. Recent studies identified HDAC4 as a critical component of several neurological processes including neuronal survival and synaptic plasticity in healthy and diseased brains. However, little is known about HDAC4 cellular process like: mechanisms governing HDAC4 cellular localization, posttranslational modification and a proteolytic cleavage, especially in the diseased brains. In addition, HDAC4 transcriptional regulation has not been studied and therefore the description of the specific transcription factors and regulatory elements driving HDAC4 expression should be carefully undertaken. The presence of an inactive deacetylase domain within the class IIa HDACs might also suggest that newly designed small molecules should be rather directed to the HDAC4 known functions, including transcription binding domain, HDAC3 interaction or proteolytic cleavage sites than toward deacetylase domain. Therefore, much more research is needed to fully describe biological function of HDAC4 in the healthy and diseased brains to be able to shape future therapeutic strategies for a various disorders.

# **REFERENCES**

nuclear pools of histone deacetylase 4. *Mol. Cell. Biol.* 27, 6718–6732. doi: 10.1128/MCB.00853-07

interaction with ATF4. *Cell. Signal.* 26, 556–563. doi: 10.1016/j.cellsig.2013. 11.026

**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.

*Received: 17 October 2014; paper pending published: 19 November 2014; accepted: 28 January 2015; published online: 24 February 2015.*

*Citation: Mielcarek M, Zielonka D, Carnemolla A, Marcinkowski JT and Guidez F (2015) HDAC4 as a potential therapeutic target in neurodegenerative diseases: a summary of recent achievements. Front. Cell. Neurosci. 9:42. doi: 10.3389/fncel. 2015.00042*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2015 Mielcarek, Zielonka, Carnemolla, Marcinkowski and Guidez. 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.*

# Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries

# **Oscar A. Carballo-Molina and Iván Velasco\***

Instituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico

#### **Edited by:**

Victoria Campos, Instituto Nacional de Neurologia y Neurocirugia, Mexico

#### **Reviewed by:**

Ertugrul Kilic, Istanbul Medipol University, Turkey Carlo Di Cristo, University of Sannio, Italy

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

Iván Velasco, Instituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, México, D.F.-04510, México e-mail<EMAIL_ADDRESS>
Damage caused to neural tissue by disease or injury frequently produces a discontinuity in the nervous system (NS). Such damage generates diverse alterations that are commonly permanent, due to the limited regeneration capacity of the adult NS, particularly the Central Nervous System (CNS). The cellular reaction to noxious stimulus leads to several events such as the formation of glial and fibrous scars, which inhibit axonal regeneration in both the CNS and the Peripheral Nervous System (PNS). Although in the PNS there is some degree of nerve regeneration, it is common that the growing axons reinnervate incorrect areas, causing mismatches. Providing a permissive substrate for axonal regeneration in combination with delivery systems for the release of molecules, which enhances axonal growth, could increase regeneration and the recovery of functions in the CNS or the PNS. Currently, there are no effective vehicles to supply growth factors or cells to the damaged/diseased NS. Hydrogels are polymers that are biodegradable, biocompatible and have the capacity to deliver a large range of molecules in situ. The inclusion of cultured neural cells into hydrogels forming three-dimensional structures allows the formation of synapses and neuronal survival. There is also evidence showing that hydrogels constitute an amenable substrate for axonal growth of endogenous or grafted cells, overcoming the presence of axonal regeneration inhibitory molecules, in both the CNS and PNS. Recent experiments suggest that hydrogels can carry and deliver several proteins relevant for improving neuronal survival and axonal growth. Although the use of hydrogels is appealing, its effectiveness is still a matter of discussion, and more results are needed to achieve consistent recovery using different parameters. This review also discusses areas of opportunity where hydrogels can be applied, in order to promote axonal regeneration of the NS.

**Keywords: axotomy, growth factors, injury response, grafting, surgical intervention**

### **INTRODUCTION**

The nervous system (NS) is responsible for the interaction between organisms and their environment; it confers the ability to respond to external stimuli. However, when an injury occurs in this system, such ability is impaired. Understanding fundamental mechanisms involved in the response to damage might be used to design therapeutic interventions aimed to promote functional recovery. Axonal regeneration capacity is very limited in the Central Nervous System (CNS; Gurgo et al., 2002; Case and Tessier-Lavigne, 2005). Although the Peripheral Nervous System (PNS) is able to grow axons after a nerve injury, the lost function is not always restored, because the regenerated axons are unable to reinnervate areas previously connected by them (Johnson et al., 2005). This review describes first the elements that impede axonal regeneration following injury in CNS and PNS, and later discuss how hydrogels might attenuate the inhibitory elements for axonal regeneration in both systems.

#### **NERVOUS SYSTEM RESPONSE TO INJURY AND ITS ROLE IN AXONAL GROWTH INHIBITION**

An injury in the NS could imply a loss of tissue, interrupted communication caused by damage of synaptic contacts or disrupted information flow between cell soma and axons. Different events occur after NS damage, depending on several factors, such as the type of injury. Many of these events are responsible for the inhibitory environment during axonal regeneration. We next describe the differential responses to lesion of CNS and PNS.

 White cells and macrophages enter through the lesion site and migrate to the surrounding neural tissue, releasing various cytokines and chemokines (Merrill and Benveniste, 1996; Donnelly and Popovich, 2008). These events lead to the activation of astrocytes, microglia and oligodendrocyte progenitor cells, to form the glial scar around the lesion site (Shearer and Fawcett, 2001; Kawano et al., 2012; **Figure 1A**). These activated cells release different molecules involved in inflammation, BBB restoration and neuroprotection (Yiu and He, 2006; Rolls et al., 2009). Glial scar isolates the damage area from adjacent tissue (**Figure 1A**); this contributes to maintaining homeostatic functions as ion and fluids balances, production of pro- and anti-inflammatory molecules, secretion of growth factors and free radicals elimination (Yiu and He, 2006; Rolls et al., 2009). In addition, a fibrotic scar (**Figure 1A**), which is produced by the intrusion of fibroblasts from the damaged meninges and that release extracellular matrix (ECM) proteins such as type IV Collagen, Fibronectin and Laminin, also forms around the site of lesion (Kawano et al., 2012). Moreover, fibroblasts and astrocytes cooperate to establish a continuous basal lamina around the glial scar (Mathewson and Berry, 1985; Shearer and Fawcett, 2001). The barrier formed by the glial and fibrous scars helps to contain the damage, preventing it from spreading and affecting surrounding tissue. The functions of these barriers are not fully understood yet, but their inhibitory effect on axonal growth has been extensively documented (Fawcett and Asher, 1999; Sandvig et al., 2004; Silver and Miller, 2004; Yiu and He, 2006; Fitch and Silver, 2008).

#### **Inhibitory elements for axonal growth**

Various elements have been described as being responsible of the adverse environment for axonal growth. The glial scar constitutes a physical barrier that impedes passage of axons across the lesion site (**Figure 1A**). In addition, the activated glial cells in the scar secrete ECM components, especially chondroitin sulphate proteoglycans (CSPGs) such as Neurocan, Brevican, Versican and NG2 (Fawcett and Asher, 1999; Tang et al., 2003), which exert an inhibitory influence on axonal growth (Shearer and Fawcett, 2001; Tang et al., 2003; Silver and Miller, 2004). Activation of Rho small GTPase proteins which are recognized by CSPGs blocks actin polymerization in growing neurites (Sandvig et al., 2004; Díaz-Martínez and Velasco, 2009). Some of the inhibitory molecules over-expressed in the site of lesion are Myelinassociated glycoprotein, Oligodendrocyte-myelin glycoprotein, Nogo protein and its receptor Nogo-66, Semaphorin 4D, Ephrin B3 (Kawano et al., 2012), Semaphorin 3D (Pasterkamp et al., 1999) and Ephrin B2 (Bundesen et al., 2003). The lesion site has been shown to secrete chemo-repulsive molecules like Tenascin (McKeon et al., 1991) and Semaphorin 3A (Pasterkamp et al., 1999). The fibrotic scar also produces inhibitory elements: it has been reported that fibroblasts express NG2 proteoglycan (Tang et al., 2003), Phosphocan (Tang et al., 2003), Tenascin-C (Tang et al., 2003), Semaphorin 3A (Pasterkamp et al., 1999) and Ephrin B2 (Bundesen et al., 2003). These data indicate that glial and fibrous scars contribute to the low rate of axonal regeneration. One option to bypass these inhibitory effects would be to prevent scar formation, but this could imply secondary effects, such as spreading of the damage. Alternatively, a modification of the lesion environment by introducing a device that is a permissive for axonal growth is feasible.

# **PNS**

The damage produce diverse signals that indicate the neuronal cell to either go into regeneration processes or to undergo programmed cell death (Maripuu et al., 2012). Ca2+ entry (Maripuu et al., 2012) and the interruption of the retrograde transport system are the initial signals of neuronal damage (Dahlin, 2008). Damage to axons also release growth factors such as Ciliary Neurotrophic Factor, Leukemia Inhibitory Factor and Interleukin-6 in the site of injury (Hanz and Fainzilber, 2006; Raivich and Makwana, 2007). After a peripheral axon is cut or crushed, the distal part of the severed axon suffers a degenerative processes (**Figure 2A**) termed Wallerian degeneration (Mietto et al., 2008; Freeman, 2014), that consists in the degradation of axonal organelles and proteins, and disintegration of axonal structures (Mietto et al., 2008). In order to obtain successful axon regeneration, activation and proliferation of Schwann cells (SC) are needed (Dahlin, 2008). SC and macrophages phagocyte the disrupted myelin sheath and cell debris (**Figure 2A**) to clear the zone (Johnson et al., 2005). In addition, SC have been shown to provide a favorable substrate for regenerating axons (Maripuu et al., 2012). As a part of the regenerative processes, several axon growth cones emerge from the proximal stump (**Figure 2A**) and their growth is guided by Bugner bands, which are SC apposed around the basement membrane (Valls-Sole et al., 2011). Basement membrane is constituted by ECM proteins such as Laminin and contributes to the adhesion and guidance of cells during development (Silver and Miller, 2004). These emerging sprouts grow to reach the distal stump across the site of lesion. However, because the adverse environment, most of these developments disperse in various directions and become abortive (Johnson et al., 2005; Valls-Sole et al., 2011; **Figure 2A**). The few sprouts that successfully reach the distal stump grow in close apposition with SC (Johnson et al., 2005). To complete this processes, the regenerated axon is myelinated by SC to produce junctional nodes of Ranvier and internodal Schmidt-Lanterman incisures (Johnson et al., 2005). After the regeneration process, SC release Nerve Growth Factor (NGF) and Glial cell-Derived Neurotrophic Factor (GDNF) in the regenerated site (Maripuu et al., 2012).

#### **Elements involved in the axonal regeneration process in the clinic**

Although the PNS has some regeneration capacity, functional restoration is not always achieved (Hill et al., 2011; Valls-Sole et al., 2011). Factors involved in the recovery are age (Lundborg and Rosén, 2001; Ruijs et al., 2005), the period of time between injury and medical assistance (Ruijs et al., 2005), the type of nerve that is damaged and the magnitude of injury (Lundborg, 2004); for example, when the lesion consists in a moderate compression, the chances of regeneration are higher (Lundborg, 2004) than when the axon has been transected (Maripuu et al., 2012). Current treatments trying to reconnect the severed nerve offer a low success rate. One major problem in these surgical procedures is that axons do not innervate the correct area, since many axons

compete to connect and this lead to loss of selectivity (Valls-Sole et al., 2011). These mismatches could convey disturbances in signaling between CNS neurons and PNS neurons, which result in sensorimotor alterations (Valls-Sole et al., 2011). Although

PNS has more permissive environment in contrast to CNS, the majority of regeneration processes do not reach to innervate the pre-lesional area (**Figure 2A**), which leads to several adverse consequences.

#### **SCAFFOLDS THAT PROMOTE AND GUIDE AXONAL GROWTH BIOMATERIALS**

Biomaterials possess properties that make them biocompatible, meaning that they do not produce cytotoxic or immunologic reactions; their components should be susceptible to modifications, and the process of its production ideally should be easy and reliable (Holmes, 2002). It is important to mention that the specific requirements will dictate the origin of biomaterials. Natural biomaterials such as collagen matrix mimic largely the extracellular environment. However, natural materials present disadvantages such as lot variability (Zhang et al., 2005b), the generation of immune reactions and they may contain pathogenic agents (Holmes, 2002). On the other hand, synthetic materials can have a more consistent quality control but are not always compatible with host tissue (Metcalfe and Ferguson, 2007). Other advantages of synthetic materials are that they are free of pathogenic agents (Holmes, 2002) and could be readily modified in order to elicit a tissue response, for example, it is possible to add the isoleucine-lysine-valine-alanine-valine (IKVAV) motif which enhances cellular adhesion (Cheng et al., 2013).

#### **SCAFFOLDS**

The purpose of implanting a biomaterial in a tissue is to provide suitable physical support to cells in order to achieve regeneration. This substrate should mimic as much as possible the natural matrix, so it should constitute a three-dimensional (3D) structure with porous size that allows the exchange of nutrients and oxygen (Hollister, 2005; Zhang et al., 2005b). Furthermore, it needs to provide a substrate for cellular adhesion, and in selected cases, could promote guided growth, proliferation, differentiation or apoptosis by scaffold-cell or by cell-cell interactions (Hollister, 2005; Zhang et al., 2005a). Some scaffolds such as hydrogels, as we will see later, have the ability to deliver several components. Therefore, the scaffold is a dynamic element that might play an important role in the regenerative process.

#### **EARLY ATTEMPTS TO PROMOTE AXONAL REGENERATIONS USING SCAFFOLDS**

The first studies, aimed to promote axonal growth after injury, were performed in the PNS using rigid materials. These attempts mainly used tubular structures to protect and guide the growth of regenerating axons. However, the used materials were not biocompatible, such as silicone conduits (Cheng and Lin, 2004), mini guide channels of a polyacrylonitrile:polyvinylchloride copolymer (Bamber et al., 1999), polytetrafluoroethylene and collagen conduits (Vasconcelos and Gay-Escoda, 2000), guidance channels of polyvinylidene fluoride (Aebischer et al., 1987), cylinders made of poly (D,L-lactic-co-glycolic acid) (Gautier et al., 1998) and poly (D,L-lactic acid) macroporous guidance scaffolds (Patist et al., 2004). Although in some cases a partial axonal regeneration was achieved, most of them did not induce successful regeneration, even after addition of trophic factors. These materials did not possess the required properties to support cell attachment, axonal growth, and some of them even induced an immune reaction. Although many biomaterials, natural or synthetic, have been proved to posses some of these properties, currently there is not a consistent strategy to induce a complete axonal regeneration either in the CNS or in the PNS.

#### **SYNTHESIS AND DEGRADATION OF HYDROGELS POLYMERIZATION**

Hydrogels are hydrophilic polymers that can incorporate up to 90% of its dry weight of water in its structure (Aurand et al., 2012; Hoffman, 2012). Water incorporation occurs during the gelling process, in which a liquid polymer solution turns into a gel structure, by polymerization of monomers (Aurand et al., 2012). Gelling involves the formation of cross-links in response to different stimuli (Sawheny et al., 1993; Aurand et al., 2012). The density of a gel can be modified, and the change in stiffness or porosity impact on the interaction of gels with cells (Lee and Mooney, 2001; Aurand et al., 2012; Kirschner and Anseth, 2013).

 The formation of gels *in situ* in living tissue limits the use of UV light, extreme pH or non-physiological temperatures. Therefore, many hydrogels have been devised to initiate the gelling process when in contact with physiological temperature (Jeong et al., 2000; Tate et al., 2001), ionic concentration (Ellis-Behnke et al., 2006; Nagai et al., 2006; Koutsopoulos and Zhang, 2012, 2013) or pH (Srividya et al., 2001; Chiu et al., 2009). The *in situ* gelling process of hydrogels is unique, because the resulting polymer can take the form of the receiving tissue. This is particularly important for some lesions of the NS, in which an irregular cavity is formed and this discontinuity impedes axonal regeneration as described previously. Hydrogels can fill completely the space, whereas preformed structures are not suitable for this application (Macaya and Spector, 2012).

#### **DEGRADATION**

Degradation of hydrogels occurs by breaking of covalent bonds (Aurand et al., 2012). Several factors influence the rate of degradation. Water access is one of them: it has been shown that when hydrogels are exposed to *in vitro* conditions, hydrolysis is the main reason of bond disruption due to high availability of water; in contrast, when hydrogels are in *in vivo* conditions, enzymatic activity, in particular metalloproteases, is the principal cause of degradation (Lutolf et al., 2003; Patterson and Hubbell, 2010). Properties of hydrogel also influence this process: in highbond density hydrogels the disruption begins from the surface, in contrast to low-bond density hydrogel, where it begins from the interior of the structure, due to the ability of water or enzymes to penetrate the hydrogel (von Burkersroda et al., 2002).

#### **HYDROGELS AS DELIVERY SYSTEMS**

Hydrogels have properties that could make them a good alternative as a drug release system. During the gelling process it is possible to incorporate different types of molecules or cells into the gel structure (Nagai et al., 2006; Kobsa and Saltzman, 2008; Censi et al., 2012; Koutsopoulos and Zhang, 2012). The incorporation of molecules into hydrogel is facilitated by the high quantity of water that permits the uptake and diffusion of soluble molecules (Nagai et al., 2006; Censi et al., 2012; Koutsopoulos and Zhang, 2012). The incorporation and release process is dictated by the characteristics of the hydrogel such as the size of porous and the molecular properties such as the monomer's molecular weight and its electrical charge (Nagai et al., 2006; Censi et al., 2012). In the case where the porous size is bigger than the molecule, the release occurs by diffusion (Amsden, 1998). On the other hand, when the molecule is larger than the porous, degradation, swelling and erosion of hydrogel permit the delivery of the molecule (Censi et al., 2012).

Administration of drugs is a necessity in the treatment of many injuries and diseases; however, commonly these drugs inside the body are metabolized and therefore have a limited time window to exert their actions. A local and controlled delivery of drugs could improve the treatments of many diseases or injuries, especially those that occur in the CNS. The delivery of drugs in the CNS implies a great challenge because the presence of BBB and the blood–spinal cord barrier, that impede the passage of many substances to the CNS (Pakulska et al., 2012). Some current delivery drugs methods into the CNS are bolus injection and catheter/minipump systems (Pakulska et al., 2012). Bolus injection into the intrathecal space is affected by the constant flow of cerebrospinal liquid, which disperse the drug, reducing its local effect (Pakulska et al., 2012). On the other hand, the use of a catheter/minipump system has high infection probabilities, due to the external minipump location. Furthermore, it is frequent that catheters suffer dislodgement, kinking, tearing and disconnection (Penn et al., 1995).

Because of the *in situ* gelling process, it is possible to use hydrogels as a local delivery system (Censi et al., 2012; Koutsopoulos and Zhang, 2012; Macaya and Spector, 2012; Pakulska et al., 2012), although it will be necessary to find out the best alternative to introduce the hydrogel into the brain or another site of the NS. It is feasible to put the hydrogel in a damaged area and release molecules there, allowing the local delivery of a drug, which could enhance the effectiveness of treatment. In addition, the use of biodegradable hydrogels is especially relevant for long-term treatments, since it will prevent repetitive invasive interventions. The data demonstrate that hydrogels are a very versatile release system because is possible to manipulate the rate of delivery and the rate of degradation. Hydrogels could be modified to release some medicament depending on the specific circumstances, like the half-life of the medicament, the dosage or the time that is required for treatment.

# **HYDROGELS PROMOTE AXONAL REGENERATION**

Hydrogels present characteristics that make them good candidates to fulfill the needs required to promote axonal regeneration after lesions of the CNS, such as filling up the cavity of a lesion with a suitable substrate (**Figure 1B**). Although the PNS environment is less restrictive for axonal growth, as described previously, there are many challenges to achieve a successful regeneration. It is essential to reduce the probability of mismatches by providing guidance cues for correct reinnervation, and hydrogels can help to attain this task (**Figure 2B**). However, a note of caution is appropriate because, in addition to act as scaffold and delivery tools, hydrogels might represent a physical barrier for both cellular and axonal reorganization. In this section we review the published evidence showing that hydrogels promote axonal regeneration both *in vitro* and *in vivo*.

#### **IN VITRO STUDIES**

Hydrogels properties such as its high water content, their porous constitution and the three dimensional (3D) networks formed during gelling, mimic to some extent the ECM found in tissues (Geckil et al., 2011; Aurand et al., 2012; Kirschner and Anseth, 2013), making possible to culture cells in 3D structures *in vitro*. These structures are closer to the *in vivo* environment than the classic two-dimensional cultures (Zhang et al., 2005b).

Early work (Holmes et al., 2000) aimed to obtain selfassembling peptides with motifs similar to the arginine-glycineaspartate (RGD) present in several ECM proteins. The authors substituted glycine with alanine (A) and repeated the RADA sequence several times. To assess the suitability of this selfassembling peptide hydrogel, a direct comparison with Matrigel (a commercial substrate containing ECM derived from carcinoma cells) was made after culturing rat hippocampal neurons. No differences in synaptic activity measured by the endocytosis marker FM1-43 were found, showing that this hydrogel can support neuronal maturation. Recently, RADA-containing peptides were used to form 3D structures to allocate neural stem progenitor cells (NSPC) in order to evaluate proliferation and neuronal differentiation (Koutsopoulos and Zhang, 2013). Both Matrigel and the self-assembled peptide-based hydrogels sustained these parameters. Matrigel was efficient during the first 2 weeks, but the hydrogel allowed neuronal survival for over 5 months. These data demonstrate that hydrogels support neuronal differentiation and long-term survival with signs of maturity.

In another study, using dopaminergic cells, Semaphorin 3A was coupled to a PEG hydrogel containing silica particles to assess the effects on axonal growth. Semaphorin 3A is a soluble protein implicated in the axonal growth of dopaminergic neurons during brain development (Hernández-Montiel et al., 2008). Application of recombinant Semaphorin 3A to these dopaminergic neurons obtained from the developing midbrain or from *in vitro* differentiated mouse embryonic stem cells caused increased growth of axons in a collagen gel system in culture. This effect was neutralized by anti-Neuropilin receptors (Tamariz et al., 2010). A significant increase in axonal length was observed with the PEG hydrogel containing either 2 or 5 µg/ml Semaphorin 3A compared to controls (Tamariz et al., 2011). Recently, a PEG hydrogel device was designed to evaluate the influence of distance in the application of a potential axonal growthpromoting molecule on murine embryonic stem cell-derived neurons. These authors conjugated Insulin-like Growth Factor 1 to poly-lactic-co-glycolic acid particles with several admixtures that resulted in different release kinetics: early, intermediate and late. The optimal conditions were: (i) a distance up to 2 mm between the poly-lactic-co-glycolic acid particles and neurons; (ii) a sequential array of early, intermediate and late release conjugates; (iii) the early release particles placed closer to the cells and those with late kinetics placed away (Lee et al., 2014).

An intermediate step between two-dimensional *in vitro* cultures and *in vivo* studies is the culture of organotypic slices, because they maintain the ECM and the 3D organization. In cultured spinal cord slices placed on different substrates such as membrane inserts, Collagen gel, soluble hyaluronic acid and hyaluronic acid-based hydrogel, different cell-type specific markers were analyzed. The hydrogel group preserved better than the other groups the characteristics of the slice: more neurons (NeuN+), a greater proportion of choline acetyltransferasepositive neurons, well-preserved astroglia and less number of activated microglial cells were reported (Schizas et al., 2014). The data of these works confirmed that hydrogels could be used as a delivery system to promote axonal growth, and preserve better the organotypic cultures. These characteristics might be useful to promote a successful axonal regeneration *in vivo*.

#### **IN VIVO STUDIES**

#### **Hydrogels as a strategy for promoting regeneration in the brain**

Several groups have assessed the biocompatibility of hydrogels in the absence of a lesion. PEG hydrogels with 13% and 20% macromer weight, and 20% PEG conjugated with GDNF were implanted into the cortex and striatum of nonhuman primates. Four months after implantation the astroglial and microglial reactions were present around the implant site of all groups, including sham. The 13% PEG hydrogel generated fewer reactions, probably due to its faster degradation (Bjugstad et al., 2008). This same group evaluated the biocompatibility of different weight percent of PEG hydrogel implanted as strands across the rat brain. The analysis in striatum revealed that both 13% and 20% hydrogels attenuate the acute response of reactive glia, compared to the sham group after 56 days (Bjugstad et al., 2010). However, when PEG was conjugated with silica particles and implanted into striatum, a higher amount of macrophages and glial cells were founded around the injection site after 30 days, compared to controls. The authors correlated this enhanced glial reaction with the presence of silica particles that were not degraded (Tamariz et al., 2011).

In some cases, one important limitation for axonal regeneration is the presence of a cavity in the damaged tissue (**Figure 1A**). Hydrogels have been proved to be able to fill such cavity and promote axonal growth (**Figure 1B**). After resection of a fraction of the cerebral cortex, the resulting cavity was filled with a hydrogel based on self-assembling peptides or with saline solution. After 6 weeks the hydrogel significantly reduced the lesion volume, and cellular ingrowth was detected. A significant decrease in astrocytic cells and macrophages was observed in the first 2 weeks, compared to the saline group (Guo et al., 2009). Hou et al. also caused a cortical damage in rats, but they used hyaluronic acid-based hydrogel, either alone or modified to incorporate Laminin in its structure, to fill the cavity. After six and twelve weeks hydrogels allow cell infiltration, angiogenesis and inhibition of the glial scar; however, only the hydrogel with Laminin was permissive for neurite growth (Hou et al., 2005).

One of the first attempts to evaluate the implantation of hydrogels together with living cells in the brain was made by Woerly et al. (1996). In this study SC, neonatal astrocytes or cells dissociated from embryonic cerebral hemispheres were entrapped in (N-(2-hydroxypropyl)-methacrylamide)-based hydrogel. Hydrogels containing SC were implanted into the rat neocortex, and promoted cellular and axonal ingrowth within the polymer. In another study, NSPC were encapsulated into self-assembling peptide hydrogels modified to include the IKVAV motif derived from Laminin (Cheng et al., 2013). Such cell-containing hydrogels were used to fill the cavity caused by a mechanical lesion in the neocortex of rats. Hydrogels made with the IKVAV motif and NSPC promoted better tissue regeneration and presented neurogenesis, compared to hydrogels without the motif, which promoted modest tissue regeneration and had prevalent glial differentiation. This study is in agreement with previous data (Hou et al., 2005) that demonstrate that the Laminin or Laminin-derived motif incorporated to hydrogel structure allow the recovery of tissue continuity. Another group tried to promote tissue recovery with a different strategy, which consisted in incorporating GDNF to gelatin-based hydrogels, with the objective to attract to the site of lesion the endogenous NSPC present in the adult subventricular zone. The hydrogel loaded with GDNF attenuated the astroglial reaction, promoted neurite growth into the site of lesion and induced the migration of neuroblasts towards the lesion site. However, cells did not reach the site of lesion, and the migration effect was observed only at 7 days post-lesion, disappearing after 21 days (Fon et al., 2014).

Probably the most important aspect in the CNS is to achieve re-connection of damaged areas, which in the long run might positively impact behavior. In a model where hamsters' optic tracts were severed and the resulting cavity was filled with a selfassembling peptide hydrogel or with saline solution, researchers observed that the hydrogel helped to reconnect the areas around the lesion after 6 weeks, in contrast to saline-treated animals. More importantly, vision was improved in the hydrogel-treated group (Ellis-Behnke et al., 2006). Although the degree of axonal re-growth varies depending on the strategy used, hydrogels have been demonstrated to be able to fill the lesion cavity with a suitable substrate for axonal growth, and the further addition of trophic factors or cells increases the possibilities of improvement after traumatic lesions in the brain (**Figure 1B**). However, in general terms, the evidence is still insufficient to say that hydrogels would substitute current treatments for brain lesions.

#### **Hydrogels promote regeneration in the spinal cord**

The studies showing that hydrogels promote axonal growth *in vitro* prompted investigators to test if implantation of hydrogels into the damaged spinal cord could promote recovery. A hyaluronic acid hydrogel was evaluated *in vitro* and *in vivo* to investigate if it was able to promote neurite growth. Although this hydrogel promoted neurite growth *in vitro*, it was not sufficient to achieve functional recovery when implanted in rats with complete transected thoracic spinal cord (Horn et al., 2007). Transection of the spinal cord in cats followed by filling of the cavity with NeuroGel hydrogel (N-(2-hydroxypropyl) methacrylamide) permitted the formation of neural tissue with myelinated axons across the damaged area, connecting both sides of the cavity, and allowing infiltration of glial cells and capillary vessels (Woerly et al., 2001). In additional work, the authors found that the hydrogel prevented scar formation and that the gliosis reaction was reduced in the interface between tissue and hydrogel (Woerly et al., 2004).

One possibility in the design of a suitable scaffold is the combination of two different types of hydrogels, to obtain a better substrate. A combined poly lactic acid (PLA) and poly (2-hydroxyethyl methacrylate, PHEMA) hydrogel was devised to obtain a degradable porous structure. This mixed hydrogel was implanted in the hemisected spinal cord of rats and demonstrated to promote axonal growth into the lesion area; moreover, animals improved in the widely used behavioral Basso, Beattie and Bresnahan scale (Pertici et al., 2014). Another strategy evaluated to bridge the two stumps after a spinal cord lesion is the use of tubular structures, which provide mechanical support for axonal regeneration. A tubular device made with poly (2-hydroxyethyl methacrylate-co-methyl methacrylate, PHEMA-MMA) hydrogel was used to join the transected spinal cord of rats and this strategy resulted in the re-establishment of tissue continuity, allowing axon regeneration with minimal scar tissue. Although the empty tubular structure promoted recovery by itself, it was proposed that filling the tubular structure with a suitable substrate could be a better option to promote recovery (Tsai et al., 2004). The same group evaluated this approach (Tsai et al., 2006), filling the tubular structure used previously with matrices of Collagen, Fibrin, Matrigel, methylcellulose or smaller PHEMA-MMA tubes. In addition, Fibrin and Collagen were supplemented with Fibroblast growth factor-1 (FGF-1) and NT-3. It was observed that almost all the matrices used promoted more axonal regeneration compared to unfilled structures. The addition of FGF-1 increased the axonal regeneration of vestibular neurons, and the addition of NT-3 decreased the total number of axons regenerating from brainstem neurons.

In addition to promote axonal regeneration, hydrogels can incorporate into its structure trophic factors and release them on site after a spinal cord lesion. PHEMA hydrogels soaked with BDNF and control PHEMA hydrogels were implanted into the hemisected spinal cords of rats. Only the BDNFcontaining hydrogel allowed axonal growth into the polymer structure (Bakshi et al., 2004). Similarly, BDNF was embedded into agarose hydrogel and implanted into hemisected spinal cords of rats. It was demonstrated that it promoted greater axonal growth in contrast to hydrogels without BDNF (Jain et al., 2006). Another trophic factor evaluated in spinal cord lesions is NT-3, which was combined to a hydrogel of acrylated PLAb-PEG-b-PLA to release it in hemisected cord of rats. Animals treated with hydrogel and NT-3 presented more axonal growth into the lesion site and improved in the behavioral parameters, in contrast to animals treated only with hydrogel (Piantino et al., 2006). Collectively, these experiments strongly suggest that

incorporation of molecules that promote axonal growth and/or cell survival increases the possibilities of recuperation after a spinal cord lesion.

As mentioned earlier, hydrogels can incorporate living cells, making grafting of cells together with hydrogel an additional strategy to promote recovery. SC and NSPC have been implanted with and self-assembling peptide-based hydrogel into transected spinal cord of rats. The ingrowth of tissue to the lesion was better in animals treated with hydrogel-embedded cells than those treated with hydrogel alone. NSPC and SC can survive, migrate, and differentiate into the site of lesion, with SC promoting greater axonal growth into damaged area (Guo et al., 2007). Another study demonstrated that when the same hydrogel was implanted with SC in a moderate spinal cord contusion model in rats, there was a reduction of astrogliosis reaction, a motor recovery and infiltration of endogenous SC to the lesion site was observed (Moradi et al., 2012).

#### **Hydrogels promote regeneration in the PNS**

Although the PNS allows some degree of regeneration, mismatches are frequent and limit its recovery. The best strategy to promote axonal regeneration in peripheral nerve injuries is the use of autografts, but this convey some problems such as donor tissue morbidity and loss of function in the tissue innervated by donor nerve (Schlosshauer et al., 2006). Some attempts have been made to substitute the autografts with variable results. Hydrogel porous tubes constructed with PHEMA-MMA were implanted into interrupted sciatic nerves. At early times the autografts were more effective as evaluated by histomorphology and electrophysiology. However, after 8 weeks the scaffold showed a bimodal recovery: 60% of animals surpassed the autografts but rest did not, probably due to tube collapse (Belkas et al., 2005). A more rigid hybrid conduit was designed with poly (3,4-ethylenedioxythiophene, PEDOT) and agarose hydrogel. This device was implanted in 10 mm peroneal nerve gaps and the regeneration was evaluated by muscle mass, contractile force measurements and nerve histomorphometry. The hybrid conduits promoted better regeneration compared to agarose-only hydrogel, but autografts presented much better results (Abidian et al., 2012).

Filling of conduits with a substrate, which allows axonal growth, could enhance the regenerative process (**Figure 2B**). An empty blood vessel filled with a self-assembling peptide hydrogel, implanted in a sciatic nerve gap of 10 mm, promoted higher numbers of growing and re-myelinated axons, more SC infiltration, less presence of lymphocytes and macrophages, greater gastrocnemius muscle recovery and better behavioral improvement, compared to empty conduits. However, the recovery was not comparable in retrograde labeling and electrophysiology, to unlesioned animals (Zhan et al., 2013). Another group developed a Keratin-based hydrogel to fill commercial tubular conduits, which improved histological characteristics such as number of blood vessels, axons per area, and axon size. Furthermore, electrophysiological features such as conduction delay and impulse amplitude were better than with the empty tubular structures, and comparable to autografts. These results were obtained in mice with a 4 mm gap in the tibial nerve (Sierpinski et al., 2008) and in rabbits with a 2–3 cm sciatic nerve break (Hill et al., 2011). The same group characterized the early cellular response after implantation of a commercial tubular structure filled with Keratin-based hydrogel, Matrigel or saline solution in rats presenting a 1 cm sciatic nerve injury. Significant differences present in the hydrogel group compared to others were: an earlier migration of dedifferentiated endogenous SC from the proximal end, faster SC dedifferentiation, higher myelin debris clearance, and decreased macrophage infiltration. However, others parameters, such as axon density, SC labeling or the amount of cells in the distal nerve did not present differences (Pace et al., 2013). It is worth mentioning that this is the only study in which the cellular response was characterized post-hydrogel implantation after peripheral nerve injury.

The release of molecules *in situ* after a lesion could enhance axonal growth through the lesion, contributing to reinnervation of correct areas (Macaya and Spector, 2012), increasing the possibilities of recovery (**Figure 2B**). Animals suffering from a 10 mm gap in the sciatic nerve were implanted with PHEMA-MMA hydrogel porous tubes, filled with Collagen matrices supplemented with NT-3, BDNF and FGF-1. The rats treated with growth factors presented better axonal regeneration compared to animals receiving empty tubes, or Collagen without factors. Tubes filled with collagen and 10 µg/ml FGF-1 presented similar number of fibers with diameters similar to animals that received autografts (Midha et al., 2003). Similarly, polysulfone tubes filled with agarose hydrogel containing Laminin-1 and NGF, implanted in the severed sciatic nerve caused equivalent recovery to animals that received autografts in parameters such as morphology of the regenerated nerve and the density of myelinated axons. However, although the functional recoveries of sciatic nerve were similar after hydrogel or autograft treatment, these values were significantly lower than the non-lesioned condition (Yu and Bellamkonda, 2003).

Ultrafiltration membrane conduits filled with a selfassembling peptide hydrogel containing SC were implanted into the damaged sciatic nerve. This device caused better axonal growth and linear alignment of nerve fibers with SC than conduits filled with: (a) self-assembling peptide-only hydrogel; (b) alginate/Fibronectin hydrogel or (c) alginate/Fibronectin with SC. (McGrath et al., 2010). These studies demonstrated that the combination of a tubular structure, which provides mechanical support, filled with hydrogels increases the possibilities of axonal regeneration after peripheral nerve injury. In addition, this system could be improved by the addition of trophic factors or cells (**Figure 2B**). The reported recoveries are to some extent similar to those resulting from autografts, the current gold standard to treat peripheral nerve damage. However, further studies that evaluate the recovery with additional parameters, such as electrophysiological studies and anterograde/retrograde labeling through regenerated axons across the damaged area are still needed.

#### **CONCLUSION**

Increasing the possibilities for axonal regeneration after neuronal damage is a complex challenge, because it is necessary to overcome several limitations, which might imply different strategies. Hydrogels have demonstrated to be useful to overcome some of these barriers, particularly by providing an adequate substrate for axonal growth. Their versatility allows modification of important parameters, which can positively impact on axonal regeneration, and this is a significant advantage compared to other biomaterials. Although different strategies such as implantation of hydrogel, alone or combined with trophic factors or with cells, have proved to promote axonal regeneration in different animal models, more research is needed to determine if hydrogels can be applied in the clinical setting in the future. Tissue regeneration seems to consistently occur after hydrogel application, but other parameters, particularly the electrophysiological and behavioral tests show more variable results, and these shortcomings will hopefully be resolved soon.

#### **ACKNOWLEDGMENTS**

The work in the laboratory of I.V. is supported by grants from Conacyt (131281) and Papiit-UNAM (IN208713). Oscar A. Carballo-Molina received a graduate fellowship from Conacyt. We thank Drs. Francisco Fernández de Miguel and Alfredo Varela-Echavarría for critical reading on early stages of this manuscript.

### **REFERENCES**

characteristics. *Proc. Natl. Acad. Sci. U S A* 100, 5413–5418. doi: 10.1073/pnas.<PHONE_NUMBER>

**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.

*Received: 21 October 2014; accepted: 09 January 2015; published online: 17 February 2015*.

*Citation: Carballo-Molina OA and Velasco I (2015) Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries. Front. Cell. Neurosci. 9:13. doi: 10.3389/fncel.2015.00013*

*This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2015 Carballo-Molina and Velasco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and 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*.

# Identification of age- and disease-related alterations in circulating miRNAs in a mouse model of Alzheimer's disease

#### *Sylvia Garza-Manero1†, Clorinda Arias 1, Federico Bermúdez-Rattoni 2, Luis Vaca3 and Angélica Zepeda1 \**

*1 Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Coyoacán, México*

*2 División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Coyoacán, México*

*3 Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Coyoacán, México*

#### *Edited by:*

*Rosalinda Guevara-Guzmán, Universidad Nacional Autónoma de México, Mexico*

#### *Reviewed by:*

*Hermona Soreq, The Hebrew University of Jerusalem, Israel Ertugrul Kilic, Istanbul Medipol University, Turkey*

#### *\*Correspondence:*

*Angélica Zepeda, Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Coyoacán, CP 04510, México e-mail: azepeda@ biomedicas.unam.mx*

#### *†Present address:*

*Sylvia Garza-Manero, Epigenetics Unit, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK* Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by the progressive decline of memory and cognition. Histopathologically, two main hallmarks have been identified in AD: amyloid-β peptide extracellular neuritic plaques and neurofibrillary tangles formed by posttranslational modified tau protein. A definitive diagnosis can only be achieved after the *post mortem* verification of the histological mentioned alterations. Therefore, the development of biomarkers that allow an early diagnosis and/or predict disease progression is imperative. The prospect of a blood-based biomarker is possible with the finding of circulating microRNAs (miRNAs), a class of small non-coding RNAs of 22–25 nucleotides length that regulate mRNA translation rate. miRNAs travel through blood and recent studies performed in potential AD cases suggest the possibility of finding pathology-associated differences in circulating miRNA levels that may serve to assist in early diagnosis of the disease. However, these studies analyzed samples at a single time-point, limiting the use of miRNAs as biomarkers in AD progression. In this study we evaluated miRNA levels in plasma samples at different time-points of the evolution of an AD-like pathology in a transgenic mouse model of the disease (3xTg-AD). We performed multiplex qRT-PCR and compared the plasmatic levels of 84 miRNAs previously associated to central nervous system development and disease. No significant differences were detected between WT and transgenic young mice. However, age-related significant changes in miRNA abundance were observed for both WT and transgenic mice, and some of these were specific for the 3xTg-AD. In agreement, variations in the levels of particular miRNAs were identified between WT and transgenic old mice thus suggesting that the age-dependent evolution of the AD-like pathology, rather than the presence and expression of the transgenes, modifies the circulating miRNA levels in the 3xTg-AD mice.

**Keywords: early diagnosis, neurodegenerative diseases, blood-based biomarker, Alzheimer models, plasma, pathological aging, prognosis, 3x-Tg**

# **INTRODUCTION**

Alzheimer's disease (AD) is the most common cause of dementia clinically characterized by the progressive decline of memory. At the histopathological level two main hallmarks define it: the abnormal extracellular accumulation of amyloid-β peptide (Aβ) into neuritic plaques and the formation of intraneuronal neurofibrillary tangles (NFTs) composed by posttranslational modified tau protein. These pathological markers are mainly located in hippocampus and cortical regions (Selkoe, 2001; Ballard et al., 2011) and are accompanied by synaptic loss early in the disease, which constitutes the major morphological correlate of memory decline (Terry et al., 1991; Dekosky et al., 1996). AD is the major neurodegenerative disorder in the elderly; the prevalence of the disease increases with age, thus aging constitutes the main risk factor (World Alzheimer Report, ADI, 2009). Two recognized forms of the disease include the sporadic AD (SAD) and the familial AD (FAD). SAD accounts for more than 99% of total cases and is characterized by a late-onset of symptomatology, whereas FAD is an early-onset form of the disease associated with mutations in three genes encoding for the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) transmitted in an autosomal dominant pattern (Goate et al., 1991; Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995).

Currently, amyloid-imaging positron emission tomography (PET) through the Pittsburgh Compound-B (PIB) tracer is the best tool for identifying amyloid deposits in patients (Klunk et al., 2004; Alkalay et al., 2013). However, a definitive diagnosis of AD can only be achieved after *post mortem* histopathological verification. Therefore, the development of sensitive and specific non-invasive methods to detect molecular markers or to best predict the disease progression is crucial. Among different prospects of a blood-based biomarker, the finding that circulating microRNAs (miRNAs) derived from different types of cells can be detected, make possible their use as non-invasive markers of brain dysfunction. miRNAs are small non-coding RNAs of 22–25 nucleotides length that posttrancriptionally regulate the level of many proteins via mRNA silencing or degradation (Bartel, 2009; Fabian and Sonenberg, 2012; Ameres and Zamore, 2013). They travel through blood within specialized vesicles known as exosomes, associated with protein complexes or into high density lipoproteins as well (Valadi et al., 2007; Arroyo et al., 2011; Vickers et al., 2011).

 Recent studies performed in plasma samples of potential AD cases suggest the possibility of finding differences between circulating miRNA levels of AD cases and control subjects (Geekiyanage et al., 2011; Kumar et al., 2013; Leidinger et al., 2013; Tan et al., 2013; Burgos et al., 2014; Kiko et al., 2014). In this regard, studies have aimed at analyzing miRNA levels at a single time-point, which may limit their use as biomarkers or as disease predictors. The possibility to evaluate circulating miRNA levels at different time-points in the progression of the disease could provide information regarding key physiopathological features of AD and could contribute in establishing early blood-based molecular biomarkers for the disease. Analyzing miRNA levels in blood samples from patients at an early time-point is hampered by the fact that symptoms do not develop until the disease is at an advanced stage. Thus, in this study we used the triple transgenic mouse model (3xTg-AD) which reproduces the age-dependent progress of amyloid pathology and develops the tauopathy similar to that found in AD (Oddo et al., 2003). In order to evaluate specific miRNA levels in plasma samples at different time-points of the evolution of the AD-like pathology, we performed multiplex qRT-PCR and compared the levels from 3xTg-AD and WT mice of 84 miRNAs previously associated to central nervous system (CNS) development (Giraldez et al., 2005; Yoo et al., 2009; Zhao et al., 2009; Edbauer et al., 2010) and diseases such as schizophrenia (Beveridge et al., 2010), Huntington disease (Johnson et al., 2008), Parkinson disease (Kim et al., 2007; Wang et al., 2008a), and AD.

No significant differences in circulating miRNA levels were detected between 3xTg-AD and WT young mice. However, agerelated significant changes in the abundance of certain plasmatic miRNAs were observed in both WT and transgenic mice. Interestingly, some of the miRNAs that changed were specific for the 3xTg-AD. In agreement, variations in the abundance of particular miRNAs were identified between WT and transgenic old mice thus suggesting that the age-dependent evolution of AD-like pathology, rather than the presence and expression of human transgenes, modifies the circulating miRNA profile in the 3xTg-AD mice.

#### **MATERIALS AND METHODS**

#### **ANIMALS**

Homozygous 3xTg-AD developed by Oddo et al. (2003) (*n* = 14) and wild-type (WT) (*n* = 13) male mice (strain 129/C57BL6) were used in the study. For developing the transgenic mice, human APP cDNA harboring the Swedish double mutation (KM670/671NL) and human tau cDNA harboring the P301L mutation were cloned into Thy1.2 expression cassette and comicroinjected into single-cells embryos harvested from homozygous PS1*M*146*V* knockin (KI) mice generated as a hybrid 129/C57BL6 background. For this study, all used 3xTg-AD mice were genotyped by identifying the three harbored human transgenes (Supplementary Figure 1). Brains from four 3xTg-AD male mice 2–3 (*n* = 2) and 14–15 (*n* = 2) months old were used for immunohistochemical analysis. Determination of circulating miRNA profiles included blood samples from six-seven animals per group and a total of four experimental groups were integrated: 2–3 months old-3xTg-AD mice (*n* = 6), 2–3 months old-WT mice (*n* = 6), 14–15 months old-3xTg-AD (*n* = 6), and 14–15 months old-WT mice (*n* = 7). The experiments were performed in accordance with local government rules and the Society for Neuroscience Guide for the Care and Use of Laboratory Animals with approval of the Animal Care Committee of the Instituto de Investigaciones Biomedicas, UNAM. Efforts were made to minimize animal suffering and to reduce the number of subjects used.

#### **GENOTYPING**

Genomic DNA was isolated from the 3xTg-AD mice tails (2–3 mm) by the *Hot-Shot* method. Tail samples were homogenized with alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA, pH 12) through mechanical action and incubated during 1 h at 95◦C. Tris-HCl buffer (40 mM, pH 5) was added to each sample for neutralization (pH 7.5). Samples were centrifuged during 2 min at 12 500 rpm, 4◦C, and the supernatant was used for PCR amplification. Human APPSwe and tauP301L transgenes were identified through their amplification products of 500 bp and 320 bp, respectively into a 2% agarose gel stained with ethidium bromide. The primers used for APP-tau PCR were 5tauRev (5 -TCCAAAGTTCACCTGATAGT-3 ), APPinternal (5 -GCTTG CACCAGTTCTGGATGG-3 ) and Thy12.4 (5 -GAGGTATTC AGTCATGTGCT-3 ). PS1M146V KI was detected by a PCR using the PS1-K13 (5 -CACACGCACACTCTGACATGCACAGGC-3 ) and PS1-K15 (5 -AGGCAGGAAGATCACGTGTTCCAAGTAC-3 ) primers, followed by an enzymatic digestion (1 h, 60◦C) with endonuclease BstEII (New England Biolabs). Products were separated into a 2% agarose gel, and identified as a 530 bp band from WT PS1, or 350 bp and 180 bp bands from mutation carriers PS1.

#### **IMMUNOHISTOCHEMISTRY**

3xTg-AD mice (*n* = 4) were anesthetized with sodium pentobarbital and transcardially perfused with 0.9% chilled saline followed by 4% chilled formaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were extracted and post-fixed during 24 h at 4◦C. Subsequently, brains were transferred to 15 and 30% sucrose solutions in 0.1 M phosphate buffer for 24 h in each concentration. 30μm-thick coronal sections were obtained in a cryostat (Microm HM550, Thermo Scientific). Free-floating sections were collected in 24-well plates (Corning) and were washed for 10, 15, and 30 min with 0.15 M PBS and 0.4% PBS-triton solutions. They were then incubated in blocking solution (0.4% PBS-triton, 3% NGS) during 2 h at room temperature, followed by an overnight incubation at 4◦C with 1:500 dilution of the primary antibody, either the MAB1560 6E10 clone (Chemicon) which recognizes aminoacids 1–17 from human Aβ peptide, or the MN1020 α-p-PHF-tau AT8 clone (Pierce, Thermo Scientific) which recognizes phosphorylated serine 202 and threonine 205 from human tau protein. Sections were washed as previously detailed and were then incubated in a 1:250 dilution of the secondary α-mouse antibody, either Alexa-488 or Alexa-555 for 2 h at room temperature. Finally, sections were washed for 10, 15, and 30 min in 0.15 M PBS, incubated for 3 min in 30 nM DAPI, and washed again. Immunofluorescence was detected using a Zeiss epifluorescence microscope under 10X and 60X.

#### **DETERMINATION OF CIRCULATING miRNA LEVELS** *Plasma collection*

Blood samples were collected from 3xTg-AD and WT mice at 2– 3 and 14–15 months of age. ∼500μL of blood were obtained from the facial vein of each mouse and collected into a new and sterile 1.5 mL centrifuge tube containing 10μL of 300 mM EDTA used as anticoagulant. Plasma was separated through two centrifugations, one at low (3000 rpm) and another at high (13,000 rpm) speed, both at 4◦C for 10 min. For determining circulating miRNA profiles, equal volumes of plasma samples from two mice within the same experimental group were pooled after plasma separation and a total of three different pools per experimental group were included in the analysis. Pooled samples were stored in ice and immediately treated following the RNA isolation protocol.

#### *RNA isolation*

RNA was isolated from each pool using the miRNeasy Serum/Plasma Kit (Qiagen) following the manufacturer instructions. Briefly, 200μL of pooled plasma were homogenized with 1000μL of Qiazol Lysis Reagent. After homogenization, 3.5μL of a 1.6 × 108 copies/μL solution of the *C. elegans* miR-39 miRNA mimic used as a spike-in control were added to each sample. Then, a phenol-chloroform extraction was performed by incubating the sample in 200μL of chloroform for 5 min at room temperature and centrifuging for 15 min at 12,000 rpm at 4◦C. The aqueous phase was mixed with 1.5 volumes of 100% ethanol and passed through the RNeasy MinElute spin column. The RNA retained in the membrane was washed with buffer solutions, 80% ethanol and eluted in 14μL of RNase free water. RNA samples were stored in ice and immediately treated for qRT-PCR protocols.

#### *qRT-PCR*

miRNA levels from plasma of 3xTg-AD and WT mice 2–3 and 14–15 months old were determined using the miScript miRNA PCR Array System (Qiagen). The System consists in three different kits: the miScript II RT Kit which performs the cDNA synthesis, the miScript SYBR Green PCR Kit which prepares the mix for qPCR reactions, and the Pathway-Focused miScript miRNA PCR Array "Neurological Development and Disease" which detects 84 different miRNAs previously associated with CNS development and disease. For the cDNA synthesis, 1.5μL of RNA isolated from plasma were used as the starting material of a 20μL-reaction that consists of a universal synthesis driven by the addition of a poly-A tail and an oligo-dT that selectively retrotranscribes the small RNA molecules (<100 nucleotides) avoiding the retrotranscription of miRNA precursors. The reaction was incubated for 1 h at 37◦C followed by a 5 min-incubation at 95◦C to inactivate the RTase enzyme. cDNA from each sample was diluted by adding 200μL of RNase free water and stored at −20◦C overnight. After thawing the cDNA on ice, 100μL of sample were added to the qPCR reaction mix using SYBR Green as a detector. 20μL of qPCR reaction mix were added to each well of the array. The multiplex qPCR was performed using the Rotor Gene 6000 under the following conditions: pre-incubation for 15 min at 95◦C, 40 denaturation-alignment-elongation (15 s at 94◦C, 30 s at 55◦C, and 30 s at 70◦C) cycles and a dissociation melt protocol.

#### **DATA ANALYSIS**

Data were analyzed using the --Ct (2−-Ct) method of relative quantification with the support of the data analysis software for miScript miRNA PCR Arrays available for users at http://pcrdataanalysis.sabiosciences.com/mirna. The settings were manually fixed at 10 cycles of baseline and 0.02 of threshold line across all PCR runs. The software calculated the -Ct value between the threshold cycle value (Ct) of each miRNA and the Ct of the *C. elegans* miR-39 miRNA mimic spike-in control used for normalization. The software also calculated the (2−-Ct) value of each miRNA between arrays from different experimental groups. Each experimental group consisted of three samples (*n* = 3) and each sample contained the pooled plasma from two animals. The mean of the relative abundance ± SD of each miRNA contained in the samples (*n* = 3) was calculated. The statistical significance between miRNAs was determined by a *t*-test. A dissociation curve analysis was performed to guarantee the presence of only one PCR product.

### **RESULTS**

In order to detect possible variations in the circulating miRNA profiles related to the AD-like pathology present in the 3xTg-AD transgenic mice, we evaluated the levels of 84 miRNAs previously associated to CNS development and disease in plasma samples of 3xTg-AD and WT mice 2–3 and 14–15 months old. As previously shown (Oddo et al., 2003), these two time-points represent different stages of evolution of the pathological features in the 3xTg-AD mice: at 2–3 months old, histopathological markers are not evidently expressed in the hippocampus whereas at 14– 15 months old they become clear in cortical and hippocampal regions. The 2–3 month old-3xTg-AD mice express human transgenes; nevertheless at this time-point we did not find extracellular amyloid aggregates in the hippocampus and scarce phosphotau (p-tau) immunoreactivity for AT8 was observed (**Figure 1A**).

As previously reported (Oddo et al., 2003) we found that the 14–15 month old-3xTg-AD mice display extracellular amyloid aggregates. Also, neuronal somata and processes were strongly immunopositive for p-tau in AD relevant residues (**Figure 1B**).

100μm (top images) and 20μm (bottom images).

#### **ABUNDANCE OF CNS DEVELOPMENT- AND DISEASE-LINKED miRNAs IN MOUSE PLASMA**

Most of the 84 evaluated miRNAs were detected in mice plasma from all analyzed groups (Supplementary Tables 1–4). Highly abundant detected miRNAs belong to the let-7 family, miR-15 family, miR-30 family, miR-24-27 cluster, miR-29 cluster, miR-17-92 cluster and its paralogs miR-106a-363 and miR-106b-25 that are characterized by their presence in multiple cell lineages as well as by their role in fundamental cellular processes. We found a group of less or not detected miRNAs such as miR-135b, miR-302a/b, miR-488, and miR-9. The plasmatic abundance of detected miRNAs was compared between groups. No significant differences were observed in the circulating miRNA profile from 2–3 months-old 3xTg-AD mice when compared to the age-matched WT group (Supplementary Table 1).

When comparing the circulating miRNA profile between the young and the old mice for both WT and 3xTg-AD, we found that aging associated with changes in the plasmatic levels of a group of miRNAs. We detected significant differences in the levels of 33 miRNAs when comparing old vs. young WT mice and in 40 miRNAs when comparing old vs. young 3xTg-AD mice; 19 of these miRNAs were common to both comparisons (**Figure 2**, Supplementary Tables 2,3). These overlapping miRNAs include family members of let-7, miR-30, miR-17-92 cluster and its paralogs. When comparing young vs. old 3xTg-AD mice, we identified a particular group of miRNAs integrated by miR-132, miR-138, miR-146a, miR-146b, miR-22, miR-24, miR-29a, miR-29c, and miR-34a which show significant differences in plasma levels only in the transgenic group, raising the possibility of age-related changes that specifically occur in the 3xTg-AD mice (**Figure 3**, Supplementary Table 3).

#### **PATHOLOGY-RELATED CHANGES IN THE RELATIVE ABUNDANCE OF PLASMATIC miRNAs**

When comparing the plasma of 14–15 months old 3xTg-AD mice vs. age-matched WT mice, we detected a significant lower abundance of miR-132, miR-138, miR-139, miR-146a, miR-146b, miR-22, miR-24, miR-29a, and miR-29c as well as a higher abundance of miR-346 (**Figure 4**, Supplementary Table 4). This finding suggests that the relative abundance of these miRNAs was altered specifically in the 3xTg-AD mice only at age 14–15 months since they were not altered at 2–3 months of age. These results correlate with the appearance of the histopathological features (**Figure 1**).

# **DISCUSSION**

AD is a neurodegenerative disorder highly related to aging that exhibits progressive manifestations at both clinical and histopathological levels. The evolution of AD neuropathological features in the 3xTg-AD is also age-dependent (Oddo et al., 2003). In this work, we evaluated the levels of circulating miRNAs in four groups of animals: young WT; young 3xTg-AD; old WT and; old 3xTg-AD. For this purpose, we pooled plasma from two different animals from the same group and conformed three samples per group. Obtaining sufficient plasma for miRNA isolation can be problematic with young animals and getting sufficient animals with the triple transgene is difficult especially with old animals, since their health declines rapidly and mortality increases. Pooling the samples on the one hand, may pose a limitation given that each sample contains plasma from two different animals. But on the other hand, this design allowed us to avoid the bias from changes in circulating miRNAs associated to each individual. Even when the 3xTg-AD already bears the pathological transgenes, we did not find changes in the circulating miRNA profile between WT and 3xTg-AD young mice. This suggests that the presence of the transgenes is not sufficient to modify the circulating levels

**and 3x-Tg-AD analyzed by qRT-PCR. (A)** The Venn diagram depicts the number of miRNAs showing statistically different plasma levels between young and old mice in WT (33 miRNAs, in light gray) and in 3xTg-AD mice (40 miRNAs, in black) as shown by a *t*-test; *p* < 0.05. From these miRNAs, 19 are common to both comparisons (dark gray). **(B)** Relative abundance among

calculated considering the difference of the Ct of each miRNA and the Ct of the miR-39 of *C. elegans* mimic (-Ct) using the formula 2−-Ct. miR-39 from *C. elegans* was used as spike-in control in all qRT-PCR experiments. Only miRNAs that showed statistical differences when comparing young and old WT or Tg subjects are shown in the graph; *t*-test; *p* < 0.05.

**FIGURE 4 | Significant differences in the abundance of plasmatic miRNAs between aging groups analyzed by qRT-PCR.** The graph depicts the relative abundance (mean ± SD) of each one of the miRNAs that show statistically significant differences (*t*-test; *p* < 0.05) in plasma levels between 14–15 months-old WT and 3xTg-AD mice. Relative abundances were calculated with the difference of the Ct of each miRNA and the Ct of the miR-39 of *C. elegans* mimics (-Ct) using the formula 2−-Ct. Determination of circulating miRNA profiles included plasma samples from six-seven animals per group and a total of four experimental groups were integrated: 2–3 months old-3xTg-AD mice (*n* = 3), 2–3 months old-WT mice (*n* = 3), 14–15 months old-3xTg-AD (*n* = 3), and 14–15 months old-WT mice (*n* = 3) (*n* = samples).

of miRNAs at early stages of the pathology. Nevertheless, we found age-related changes in the plasmatic abundance of certain miRNAs in both WT and 3xTg-AD mice. Remarkably we found changes in specific circulating miRNAs in the aging AD model. In agreement with other studies (Grillari et al., 2010; Kato et al., 2011; Martinez et al., 2011) we found a decrease in abundance of members of the family of let-7, miR-30, miR-17-92 cluster and its paralogs miR-106a-363 and miR-106b-25 in WT and 3x-Tg-AD aged mice. Let-7 family is highly involved in developmental processes in multiple species; it is required for cell differentiation thus its levels increase in late developmental stages and remain high in the adult (Thomson et al., 2004). Accordingly, let-7 family members target important cell cycle molecules (Schultz et al., 2008). miR-17-92 cluster and its paralogs are other important cell cycle modulators; interestingly, they are up-regulated in cancer and suppression of these clusters' members induce cell growth arrest in cancer models, whereas they are down-regulated in aging and their overexpression induces cellular senescence (Grillari et al., 2010).

In addition, we found some miRNAs involved with an inflammatory response such as miR-146a and miR-146b that display altered levels in plasma of both groups of old mice. This is particularly relevant considering that it has been established that inflammation is a condition closely related to the aging process. miR-146a and 146b are NF-κB sensitive-miRNAs that suppress the immune response by inhibiting the pro-inflamatory immune cell signaling (Taganov et al., 2006). Interestingly, miR-146a displays a higher abundance in the 14–15-month old WT and a lower abundance in the 14–15-month 3xTg-AD as compared to their respective group of young mice. These bidirectional modulation agrees with recent data indicating that miR-146a is elevated in senescence models (Olivieri et al., 2013; Rippo et al., 2014), while it is decreased in AD (Kiko et al., 2014; Müller et al., 2014). A similar case is miR-34a, which is present with lower abundance in the 14–15-month 3xTg-AD mice as compared to the 2–3-month 3xTg-AD while showing a trend to increase with age in the WT. Higher levels of this miRNA are found in the brain, blood cells and plasma of old mice (Li et al., 2011) but the loss of miR-34a induces neurodegenerative phenotypes in *Drosophila*, and its overexpression induces longevity (Liu et al., 2012). Remarkably, lower levels of miR-34a have been reported in AD cases (Kiko et al., 2014; Müller et al., 2014).

Thus, it is evident that the age-dependent evolution of AD-like pathology produces specific changes in the circulating miRNA profile of the 3xTg-AD mice. As a consequence, we identified differences in the abundance of 10 miRNAs when comparing plasma samples of the 14–15- month 3xTg-AD with the 14–15-month WT mice. Most of the alterations observed in the aged 3xTg-AD mice consist on the reduction of plasmatic miRNAs levels as compared to the aged WT. This is in agreement with previous reports from brain, CSF and serum/plasma samples of AD patients where miR-15, miR-29, miR-101, miR-106, miR-107, and miR-181 have been shown to be diminished (Cogswell et al., 2008; Hébert et al., 2008; Wang et al., 2008b, 2011; Geekiyanage and Chan, 2011; Geekiyanage et al., 2011; Kumar et al., 2013; Tan et al., 2013; Burgos et al., 2014; Kiko et al., 2014).

**Table 1 | AD-like pathology-related miRNAs identified in this study and previously associated with AD.**

*The table contains data obtained from this study (left) and collected from others (right). It indicates the miRNA, the sample used for determination, the abundance respect to the control cases and the reference of the other studies. It also contains the mRNA-target for the miRNA: p250-GAP, a brain-enriched GTPase-activating protein for Rho Family GTPases involved in the N-Methyl-d-aspartate receptor (NMDAR) signaling; APT1, acyl protein thioesterase 1, an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse; IRAK1, interleukin-1 receptor-associated kinase 1, a kinase that associates with the interleukin-1 receptor upon stimulation which is responsible for interleukin-1 transcription of NFkB; TRAF6, TNF receptor associated factor 6 involved in the regulation of inflammation response and apoptosis; BACE1,* β*-secretase, a protease that cleaves the APP in the* β *site to produce the A*β *peptide in the amyloidogenic APP processing.*

Some of the 10 AD-like pathology-related miRNAs identified in this study have been previously associated with AD in different studies (**Table 1**). For instance, the miR-29 cluster is decreased in brain samples of AD patients, which correlates with an increase of the levels of the β-sercretase protein. Furthermore, members of this cluster have been shown to regulate the levels of the β-secretase *in vitro* (Hébert et al., 2008). Levels of miR-146a and miR-146b have been reported diminished in brain, CFS and plasma of AD patients (Cogswell et al., 2008; Kiko et al., 2014; Müller et al., 2014). These miRNAs are related to the inflammatory response, and may constitute an interesting result since neuroinflammation is a prevalent and early pathological feature of AD (Lukiw et al., 2008). In agreement with our results, lower levels of miR-132, miR-138 y miR-139 have been reported in AD brain and CFS samples (Cogswell et al., 2008; Burgos et al., 2014). miR-132 has been reported to have an anti-inflammatory effect, blocking acetylcholinesterase, elevating acetylcholine levels and consequently blocking NF-kB (Shaked et al., 2009). In neurons, miR-132 and miR-138 are expressed in response to synaptic activity and have a role in the modulation of morphologic events of neuroplasticity taking place in memory and cognition processes (Wayman et al., 2008; Siegel et al., 2009; Edbauer et al., 2010; Impey et al., 2010; Hansen et al., 2012; Bicker et al., 2014). Synapses represent vulnerable structures in AD and several pathologic molecular events take place in these sites comprising their function (Selkoe, 2002). Since memory and cognition are highly impaired in AD, the lower abundance of miR-132 and miR-138 result very interesting for analyzing the evolution of AD at a molecular level. It would be therefore interesting to determine changes in plasmatic miRNAs with the status of patients already showing mild cognitive impairment in order to advance the early diagnosis of AD.

In this work we used a pre-designed miRNA microarray platform, which included a restricted number of miRNAs previously associated to different neuropathologies. Other miRNAs not included in this design may be associated also to AD. However, and since to our knowledge this is the first study of circulating miRNAs in this transgenic model, we decided to incorporate miR-NAs that have been already associated to several neuropathologies. It is clear that given the multifactorial nature of the disease, it is likely that not only a single biomarker will meet the needs for clinical diagnosis. Here we propose that a relative simple, non-invasive procedure may provide useful information about the AD pathophysiology, detection, and evolution. Thus, combining a panel of miRNA detection with additional biomarkers may increase the sensitivity and specificity for early AD diagnosis.

#### **CONCLUSION**

The aim of this study was to identify changes in the circulating miRNA profile of a transgenic mouse model of AD in two different stages of the evolution of AD-like pathology. Using these two time-windows it was not possible to detect early modifications in miRNA levels associated with prodromal AD. However, we distinguish variations in different miRNAs once the AD-like pathology is established which advances the molecular biomarkers field. Most of the miRNAs we found as potentially interesting biomarkers for AD have no identified targets that may be assoiciated to the disease and their role in the pathological mechanisms taking place in AD remains unknown. Our results however shed light on subtle molecular modifications associated to pathological aging and open new venues for studying the role of particular circulating miRNAs in the evolution of AD.

#### **ACKNOWLEDGMENTS**

We thank Perla Moreno-Castilla, Patricia Ferrera, and Gonzalo Acero for technical assistance. This work was supported by grants from CONACyT-176589 and PAPIIT-IA200312 to AZ, CONACyT-127822 to LV, CONACyT-155242 and DGAPA-IN209413 to FB-R. SG-M received an MSc degree fellowship from CONACyT. Additional support was provided by the Program "Posgrado en Ciencias Bioquímicas" at the Universidad Nacional Autónoma de México (UNAM).

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fncel.2015. 00053/abstract

# **REFERENCES**

FMRP-associated microRNAs miR-125b and miR-132. *Neuron* 65, 373–384. doi: 10.1016/j.neuron.2010.01.005

down-regulating p250GAP. *Proc. Natl. Acad. Sci. U.S.A.* 105, 9093–9098. doi: 10.1073/pnas.0803072105

**Conflict of Interest Statement:** The Guest Associate Editor Rosalinda Guevara-Guzmán declares that, despite being affiliated to the same institution as authors Sylvia Garza-Manero, Clorinda Arias, Federico Bermúdez-Rattoni, Luis Vaca and Angélica Zepeda, the review process was handled objectively and no conflict of interest exists. 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.

*Received: 17 October 2014; accepted: 03 February 2015; published online: 19 February 2015.*

*Citation: Garza-Manero S, Arias C, Bermúdez-Rattoni F, Vaca L and Zepeda A (2015) Identification of age- and disease-related alterations in circulating miRNAs in a mouse model of Alzheimer's disease. Front. Cell. Neurosci. 9:53. doi: 10.3389/fncel. 2015.00053*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2015 Garza-Manero, Arias, Bermúdez-Rattoni, Vaca and Zepeda. 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.*

# Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

*José Correa-Basurto1, Roberto I. Cuevas-Hernández1, Bryan V. Phillips-Farfán2, Marlet Martínez-Archundia1, Antonio Romo-Mancillas3, Gema L. Ramírez-Salinas1, Óscar A. Pérez-González2, José Trujillo-Ferrara1 and Julieta G. Mendoza-Torreblanca2\**

*1 Laboratorio de Modelado Molecular y Diseño de fármacos, Departamento de Bioquímica de la Escuela Superior de Medicina del Instituto Politécnico Nacional, México City, Mexico, 2 Laboratorio de Nutrición Experimental, Laboratorio de Oncología Experimental and Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, México City, Mexico, 3 División de Estudios de Posgrado, Facultad de Química, Universidad Autónoma de Querétaro, Santiago de Querétaro, Mexico*

#### *Edited by:*

*Victoria Campos-Peña, Instituto Nacional de Neurologia y Neurocirugia, Mexico*

#### *Reviewed by:*

*Benjamín Florán, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico Alan Talevi, National University of La Plata, Argentina*

#### *\*Correspondence:*

*Julieta G. Mendoza-Torreblanca, Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, Insurgentes Sur No. 3700-C, Colonia Insurgentes Cuicuilco, Delegación Coyoacán, México City 04530, Mexico<EMAIL_ADDRESS>
> *Received: 06 November 2014 Accepted: 17 March 2015 Published: 10 April 2015*

#### *Citation:*

*Correa-Basurto J, Cuevas-Hernández RI, Phillips-Farfán BV, Martínez-Archundia M, Romo-Mancillas A, Ramírez-Salinas GL, Pérez-González OA, Trujillo-Ferrara J and Mendoza-Torreblanca JG (2015) Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations. Front. Cell. Neurosci. 9:125. doi: 10.3389/fncel.2015.00125* Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an *in silico* study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

#### Keywords: SV2A, epilepsy, levetiracetam, brivaracetam, seletracetam

# Introduction

Epilepsy is the most common chronic brain disorder that affects people of all ages. More than 50 million people worldwide have epilepsy (WHO, 2014). It is characterized by recurrent seizures,

**Abbreviations:** AM1, Austin Model 1; BRIV, brivaracetam; CNS, central nervous system; H-bonds, hydrogen bonds; -G, free energy; LEV, levetiracetam; MFS, mayor facilitator family; MDS, molecular dynamics simulation; OPLS-AA, optimized potentials for liquid simulations all atoms; PET, positron emission tomography; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Rg, radius of gyration; RMSD, root mean square deviation; RMSF, root mean square fluctuations; SAS, solvent accessible surface area; SD, standard deviation; SEL, seletracetam; SV2A, synaptic vesicle protein 2A; 3D, three-dimensional; TM, trans-membrane; Uniprot, universal protein resource; VMD, visual molecular dynamics.

which may vary from a brief lapse of attention or muscle jerks, to severe and prolonged convulsions (WHO, 2014). In most patients with epilepsy, seizures respond to available medications. However, a significant number of patients -especially in the setting of medical-intractable epilepsies- may experience different degrees of memory or cognitive impairment, behavioral abnormalities or psychiatric symptoms, which limit their daily functioning. As a result, in many patients, epilepsy may resemble a neurodegenerative disease (Ono and Galanopoulou, 2012). Epileptic seizures and/or epileptogenesis may functionally alter brain regions involved in cognitive processing, contributing to the progressive nature of epilepsy; additionally neurodegenerative cellular mechanisms may also participate. To cure epilepsy, both epileptogenesis and the associated neuro-degeneration have to be stopped and, if possible, reversed. This will require early detection through biomarkers that can reliably predict disease progression (Ono and Galanopoulou, 2012).

Synaptic vesicle protein 2A is an integral membrane protein present on all synaptic vesicles, being nearly ubiquitous in the CNS (Lynch et al., 2004). The existing evidence suggests that SV2A is critical for the proper function of the CNS. It regulates vesicle exocytosis by modulating the concentration of pre-synaptic calcium (Janz et al., 1999; Chang and Sudhof, 2009; Wan et al., 2010); maybe by binding to synaptotagmin 1 (Schivell et al., 1996, 2005; Yao et al., 2010), a calcium sensor protein implicated in synaptic vesicle exocytosis. In addition, it has been proposed that SV2A may control the release of neurotransmitters as a gel matrix or could transport ions, such as chloride or calcium (Bajjalieh et al., 1994; Janz et al., 1999; Reigada et al., 2003; Mendoza-Torreblanca et al., 2013). The SV2A protein has been associated to the physiopathology of epilepsy; newborn mice lacking SV2A experience severe seizures and die within 3 weeks, suggesting multiple neural alterations (Crowder et al., 1999; Janz et al., 1999). Moreover, SV2A protein expression is significantly reduced in brain tissue obtained from epileptic patients and in rats during epileptogenesis, suggesting that decreased SV2A contributes to the progression of epilepsy (Feng et al., 2009; Toering et al., 2009; van Vliet et al., 2009). SV2A is the molecular target of the second-generation antiepileptic drug LEV and its structural analogs BRIV, SEL and UCB-30889 (Gillard et al., 2003; Lynch et al., 2004; Frycia et al., 2010). Although LEV may acutely modulate ion channels and other targets; chronically, LEV may lead to decreased transmitter release predominantly by binding to SV2A (Surges et al., 2008; Crepeau and Treiman, 2010; Lyseng-Williamson, 2011; Deshpande and Delorenzo, 2014).

Knowing the *in vivo* expression pattern of SV2A in epilepsy patients may be essential for evaluating increased epileptogenicity and thus disease progression (van Vliet et al., 2009). Moreover, SV2A expression could also be useful as a biomarker for treatment response (de Groot et al., 2011). For this purpose, sensitive neuroimaging methods such as PET may enhance our ability to detect SV2A expression (Ono and Galanopoulou, 2012). Because PET requires the use of a radio-ligand to label the protein target, as a first approximation to synthesize a radio-ligand with a high affinity and specificity, we investigated the specific binding site(s) for racetams in SV2A and the residues involved in their interaction. Therefore, an *in silico* study was performed to identify the binding site for LEV and other racetams within SV2A. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, it was necessary to generate and validate a 3D model of this protein. This model was refined by a MDS and docking studies were performed to decipher the interactions between the racetams and SV2A.

# Materials and Methods

## SV2A modeling

The SV2A protein sequence was retrieved from the National Center for Biotechnology Information (NP\_476558.2) and UniProt (Q02563). There was no difference between these databases; the protein sequence was thus used as an input for the I-Tasser server (Roy et al., 2010); which provided a cluster of five 3D models for this protein. The Rampage server was used to obtain the Ramachandran plot, which shows the ϕ and ψ torsion angles for all protein residues. Additionally, the CPHmodels-3.2 and 3D-JIGSAWv3.0 servers were used to confirm the reproducibility of the SV2A 3D model provided by I-Tasser.

### System Preparation for Molecular Dynamics Simulation

The model of the SV2A protein was inserted into a preequilibrated membrane consisting of POPC molecules. The resulting system was then placed in a hexagonal prism shaped box with its symmetry axis, which was perpendicular to the plane of the bilayer membrane, in the *z* direction. The system was then solvated using simple point charge model water molecules. The geometry and size of the simulation box were carefully selected to reduce to a minimum the number of water molecules and thus decrease the computation time. In order to remove solvent molecules accidentally introduced in the hydrophobic region of the lipid bilayer, 19 Na+ ions were added to neutralize the whole system.