Antisense (AS) transcripts are RNA molecules that are transcribed from the opposite strand to sense (S) genes forming S/AS pairs. The most prominent configuration is when a lncRNA is antisense to a protein coding gene. Increasing evidences prove that antisense transcription may control sense gene expression acting at distinct regulatory levels. However, its contribution to brain function and neurodegenerative diseases remains unclear. We have recently identified AS Uchl1 as an antisense to the mouse Ubiquitin carboxy-terminal hydrolase L1 (*Uchl1*) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5. This is mutated in rare cases of early-onset familial Parkinson's Disease (PD) and loss of UCHL1 activity has been reported in many neurodegenerative diseases. Importantly, manipulation of UchL1 expression has been proposed as tool for therapeutic intervention. AS Uchl1 induces UchL1 expression by increasing its translation. It is the representative member of SINEUPs (SINEB2 sequence to UP-regulate translation), a new functional class of natural antisense lncRNAs that activate translation of their sense genes. Here we take advantage of FANTOM5 dataset to identify the transcription start sites associated to S/AS pair at Uchl1 locus. We show that AS Uchl1 expression is under the regulation of Nurr1, a major transcription factor involved in dopaminergic cells' differentiation and maintenance. Furthermore, AS Uch1 RNA levels are strongly down-regulated in neurochemical models of PD *in vitro* and *in vivo*. This work positions AS Uchl1 RNA as a component of Nurr1-dependent gene network and target of cellular stress extending our understanding on the role of antisense transcription in the brain.

Keywords: antisense transcription, long non-coding RNA, Parkinson's disease, Nurr1, dopaminergic cells

# Introduction

Large genomic projects such as ENCODE (Derrien et al., 2012) and FANTOM (Forrest et al., 2014) have shown that the majority of the mammalian genome is transcribed, thus generating a previously underestimated complexity in gene regulatory networks. A vast repertory of different classes of transcripts includes non-coding RNAs and RNAs of Transposable Elements (TEs), such as LINE (long interspersed nuclear element) and SINEs (short interspersed nuclear element) (Katayama et al., 2005; Faulkner et al., 2009; Kapranov et al., 2010; Fort et al., 2014). Long non-coding RNA (lncRNA) genes seem to represent the majority of cellular transcriptional output. The FANTOM project has cataloged more than 30,000 putative lncRNA transcripts by full length cDNA cloning (Katayama et al., 2005) while NON-CODEv4 currently contain 46,475 lncRNA genes (Xie et al., 2014; Quek et al., 2015).

Antisense (AS) transcripts are RNA molecules that are transcribed from the opposite strand to sense (S) genes forming S/AS pairs. These are estimated to include the large majority of protein encoding genes and about one third of lncRNAs (Chen et al., 2004; Katayama et al., 2005; Engstrom et al., 2006; Derrien et al., 2012). The most prominent class of S/AS pair is when a proteinencoding gene presents a lncRNA on the opposite strand. In a growing number of cases, AS lncRNAs have been proved to be required for proper regulation of sense genes, carrying genetic information acting at distinct regulatory levels (Yu et al., 2008; Spigoni et al., 2010; Tripathi et al., 2010). Understanding their mode of action may be also relevant for gene expression manipulation in vivo since lncRNAs may become in the near future a new class of RNA-based drugs for therapeutic intervention.

The contribution of AS lncRNAs to neurodegenerative diseases is still unclear although some significant examples in Alzheimer's disease (AD) (Faghihi et al., 2008, 2010), spinocerebellar ataxia type 7 (Sopher et al., 2011) and Huntington's disease (Chung et al., 2011) may suggest they play a prominent role in neuronal homeostasis and dysfunction.

Parkinson's disease (PD) is a slowly progressive degenerative disorder of the central nervous system (CNS) that is classically defined in terms of motor symptoms. The neuropathological hallmark in post-mortem brains is the selective degeneration of specific subsets of mesencephalic dopaminergic (DA) cells and the formation of cytoplasmic aggregates called Lewy bodies. The current model of toxicity of DA neurons includes mitochondrial dysfunction, oxidative stress and alterations in protein turnover. This stems from the observation on post-mortem PD brains as well as from the identification of genes associated to rare forms of early-onset familial PD. Some of these features are recapitulated in a neurochemical model of the disease that takes advantage of the selective accumulation of toxic MPP+ species in DA neurons.

So far, relevant examples for lncRNAs antisense to genes involved in PD have been restricted to a transcript associated to PINK1/PARK6 locus (Scheele et al., 2007).

Recently, we have identified a lncRNA that is antisense to the mouse Ubiquitin carboxy-terminal hydrolase L1 (Uchl1) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5 (Carrieri et al., 2012).

Uchl1 encodes for one of the most abundant proteins in the brain. It acts as deubiquinating enzyme, ubiquitin ligase or monoubiquitin stabilizer, thus regulating ubiquitin turnover (Liu et al., 2002). Dysfunction in UCHL1 has been reported in many neurodegenerative diseases. A missense mutation in UCHL1/PARK5 has been associated to rare cases of early-onset familial PD. Inactivating oxidative modifications of UchL1 protein have been reported in PD post-mortem brains where it correlates with the formation of protein aggregates (Choi et al., 2004; Barrachina et al., 2006; Gong et al., 2006). In this context several evidences position UchL1 as a major regulator of α-synuclein degradation and toxicity (Liu et al., 2009). Lack of hydrolase activity has also been found in recessive cases of a childhoodonset progressive neurodegeneration (Bilguvar et al., 2013). An in-frame deletion in the Uchl1 gene, as observed in the gracile axonal dystrophy mice, leads to axonal dystrophy and premature death (Saigoh et al., 1999). Reduced UCHL1 protein levels were also found in sporadic AD brains. Recently, UCHL1 overexpression has been shown to accelerate lysosomal degradation of APP, inhibit plaque formation and improve memory deficits in AD transgenic model mice (Gong et al., 2006). These data proves UchL1 activity is required for proper brain function. Furthermore, they suggest that increasing UCHL1 expression in vivo may be a safe and effective disease-modifying strategy to treat neurodegenerative diseases. It is therefore important dissecting all the molecular events involved in Uchl1 gene regulation.

AS Uchl1 is a 5′ head to head, 1.2 kb long transcript that initiates within the second intron of Uchl1 and overlaps the first 73 nts of the sense mRNA including the AUG codon. The nonoverlapping part of the transcript also contains an embedded repetitive sequence SINEB2 of the B3 subclass in the inverted orientation. AS Uchl1 is expressed in mouse mesencephalic DA neurons, the site of degeneration in PD. In physiological conditions AS Uchl1 RNA is nuclear-enriched. Upon rapamycin, it shuttles from the nucleus to the cytoplasm and specifically targets Uchl1 mRNA to heavy polysomes for translation (Carrieri et al., 2012). AS Uchl1 is the representative member of SINEUPs (SINEB2 sequence to UP-regulate translation), a new functional class of natural antisense lncRNAs that activate translation of their sense genes (Zucchelli et al., submitted).

Cap Analysis of Gene Expression (CAGE) is a technology based on the generation of short sequence tags from the 5′ end of full-length cDNAs followed by high-throughput sequencing. When mapped to a reference genome, CAGE tags survey transcription start site (TSS) activity of specific promoters and measure expression levels on a massive scale (Gustincich et al., 2003; Shiraki et al., 2003; Carninci et al., 2006). The FANTOM5 (Functional Annotation of Mammals 5) project has developed a simplified CAGE protocol adapted to single-molecule HeliScope sequencer (hCAGE) (Kanamori-Katayama et al., 2011) to decrease PCR biases and improve depth of sequencing. hCAGE technology was applied to a wide range of human and mouse tissues providing an unprecedented dataset for promoter usage analysis (Forrest et al., 2014).

**Abbreviations:** Uchl1, Ubiquitin Carboxy-terminal Hydrolase L1; S, sense; AS, antisense; hCAGE, Heliscope Cap Analysis of Gene Expression; TSS, transcription start site; lncRNA, long non-coding RNA; PD, Parkinson's disease; DA, dopaminergic; TH, tyrosine hydroxylase; Vmat2, vescicular monoamine transporter 2; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine.

Here we take advantage of FANTOM5 dataset to map TSSs and analyze the expression of the S/AS pair at the Uchl1 mouse locus. This led to the identification in the AS Uchl1 promoter region of a binding site for Nurr1, a transcription factor required for DA cells differentiation. Chromatin immuno-precipitation and quantitative RT-PCR proved that AS Uchl1 expression is under the control of Nurr1 activity. Finally, we show that transcription of S/AS Uchl1 RNA is regulated in neurochemical models of PD in vitro and in vivo.

# Materials and Methods

# Cell Lines

Murine dopaminergic MN9D cells with doxyciclin- inducible induction of Nurr1 transcription factor (MN9D Nurr1 Tet-ON stable cells, or iMN9D cells) were maintained in culture as previously described (Hermanson et al., 2003). Nurr1 expression was obtained by culture with 2.5µg/ml doxycycline for 12 h or longer, as required. Forin vitro neurochemical model of PD, iMN9D cells were treated with 100µM MPP+ for 16 h.

# ChIP Assay

Chromatin immunoprecipitation (ChIP) was performed with magnetic beads (Dynabeads, Invitrogen) following the protocol as described (Schmidt et al., 2009). For each ChIP, one confluent 100 mm plate of iMN9D cell was treated with doxycicline 2.5µg/ml. Nurr1 expression upon doxycicline treatment was followed by western blot. 1µg of ChIP-grade anti-Nurr1 antibody was used (sc-990 X). Rabbit IgG were used as negative control (Cell signaling #2729).

qPCR was performed with primers for DNA binding regions of indicated targets and distal primers were designed for an unrelated region 6000 bps upstream the AS Uchl TSS:

VMAT NBRE F: 5′ -ATTGTGCTAACATTTATTCCAGAG-3′ VMAT NBRE R: 5′ -AGGGCTTCCTACGTGACC-3′ OCN NBRE F: 5′ -CCACAACACGCATCCTTT-3′ OCN NBRE R: GGACTTGTCTGTTCTGCA-3′ AS Uchl1 NBRE F: 5′ CTTCCCATACAGCTTAGTTCC-3′ AS Uchl1 NBRE R 5′ -TTGCGTCTCTGCCAGATG-3′ Distal F 5′ -TCATCCAGCCACAAGGTCAGAG-3′ Distal R 5′ - CCAGCAGGCACACTGTTGAAC-3′ Enrichment of chromatin binding was calculated relative to

total input, as described previously (Guccione et al., 2006).

# RNA Isolation, Reverse Transcription and Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from iMN9D cells or dissected mouse ventral midbrain using TRIZOL reagent (Invitrogen) and following manufacturer's instructions. RNA was treated with DNAse I (Ambion) before use. Single strand cDNA was prepared from 1µg of purified RNA using the iSCRIPT™ cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. qRT-PCR reaction was performed using SYBR-Green PCR Master Mix (Applied Biosystem) and an iCycler IQ Real time PCR System (Bio-Rad). Oligonucleotide sequences of primers used in this study were previously described (Carrieri et al., 2012). qRT-PCR for Nur77 was performed with the following primers:

# Nur77-FWD CCTCATCACTGATCGACACG Nur77-REV CCTCCAACTTGAGGCAAAAG

# MPTP Treatment

Mice used in this study were treated according to the NIH guidelines for Care and Use of Laboratory Animals. MPTP use and safety precautions were as described previously (Jackson-Lewis and Przedborski, 2007). All animal experiments were performed in accordance with European guidelines for animal care and following SISSA Ethical Committee permissions. Mice were housed and bred in SISSA non-SPF animal facility, with 12 h dark/light cycles and controlled temperature and humidity. Mice had ad libitum access to food and water.

Eight-week-old, male, TH-GFP mice (Sawamoto et al., 2001) were subjected to a sub-acute MPTP regimen (Jackson-Lewis and Przedborski, 2007). Animals received one intra-peritoneal injection of MPTP-HCl (free base suspended in saline; Sigma-Aldrich) at 20 mg kg−1 dose every 2 h for a total of four doses over an 8 h period (Tatton and Kish, 1997; Miller et al., 2004; Gibrat et al., 2009). Injection of saline solution was used in control mice. Animals were sacrificed 2 days or 7 days after last injection, as these time-points were previously associated to variations in Uchl1 and Nurr1 expression (Miller et al., 2004; Gibrat et al., 2009). 300 DA neurons were purified by laser capture microdissection as described previously (Biagioli et al., 2009; Carrieri et al., 2012) and used for RNA extraction and qRT-PCR.

## Bioinformatic Analysis

Analysis of FANTOM5 collection of mouse libraries was performed using the Zenbu browser genomic tool (Severin et al., 2014) and publicly available FANTOM5 datasets (http://fantom. gsc.riken.jp/5/) (Forrest et al., 2014). A specific script was designed to extract expression values from graphical tables in Zenbu Genome Browser and convert into Excel spreadsheet for further analysis (Paolo Vatta, unpublished). Expression values for S/AS Uchl1 were calculated for a window of about ±800 base pairs around main TSS. Selection of brain-specific libraries was done by manual annotation.

For co-expression analysis, average expression values were calculated for S/AS transcripts in libraries that express both and divergence from average was considered.

For ChIP experiments, identification of NGFI-B binding elements was performed with the Genomatrix program (http:// genomatrix.de) and the TRANSFAC database (Knuppel et al., 1994). The mouse AS Uchl1 genome region from kb −3000 to +1000 was the reference sequence. Trascriptional binding factor motifs were chosen on the basis of core similarity (score 1.0) and matrix similarity (above 0.80).

# Results

# Evidence of AS Uchl1 Transcription in Mouse FANTOM5 Collection of Cell Lines, Primary Cells and Tissues

We first interrogated FANTOM5 expression data for almost 400 mouse samples, covering cell lines, primary cells and tissues. These dataset are build from hCAGE libraries and are based on sequencing cDNA copies of the 5′ ends of mRNAs, of which the integrity is inferred by the presence of their cap. These sequences—referred to as tags—are sufficiently long to be aligned in most cases at a single location in the genome. The first position of this alignment identifies a base pair where transcription is initiated defining a TSS. The number of times a given tag is represented in a library gives an estimate of the expression level of the corresponding transcript.

Since antisense lncRNAs are typically expressed at much lower level than overlapping protein-coding transcripts (Derrien et al., 2012; Forrest et al., 2014) (Zucchelli et al., FAN-TOM5 satellite, submitted), we used FANTOM5 mouse datasets in which no expression cutoff was applied to detect values as low as 1 count per library. Tags were positively scored if mapping to a region of about 800 bp around the main TSS of Uchl1 (**Figures 1A**, **3**). Expression was measured as Tag Per Million (TPM).

While sense mRNA is expressed in almost all analyzed samples (90% of libraries), only 40% of them score positive for antisense transcription proving AS Uchl1 expression is relatively restricted (**Figure 1B**). Sense mRNA is present at higher levels as compared to its antisense (**Figure 1C**). Both transcripts are highly expressed in the brain (**Figure 1D**). In particular AS Uchl1 RNA is present in the cortex, striatum and hippocampus (TPM 3.2- 2.2) although its highest level is measured in testis (TPM 5.38). Within FANTOM5 dataset, about 50 libraries were prepared from blood-derived primary cells (B cells, T cells, macrophages,

lymphoid and myeloid progenitor cells, megakaryocyte precursor cells) and tissues related to immune system (spleen and thymus). Very low expression is detected for AS Uchl1 (0.2 TPM) in only one/two of the replicas from naïve CD4 T cells, c-Kit+ stem cells and common myeloid progenitors (data not shown).

To assess whether transcription in opposite orientation is coregulated, we analyzed FANTOM5 libraries containing tags for both RNAs (N = 144 libraries). Since Spearman Correlation analysis could not be applied, we calculated average TPM levels for each transcript and set this value to 1. We then normalized expression values in all libraries to the reference and monitored variation from the average. We found that the majority of libraries (N = 122) displays concordant variation of S/AS transcription from the average, with both positive and negative co-regulation. When expression of S/AS pair is not co-regulated (N = 22), variation from average values goes in both directions and is almost equally present in non-brain (N = 9) and brain (N = 13) libraries.

 As previously found, sense and antisense levels are positively co-regulated with values in neonatal tissues often higher than in adult samples of the same area (**Figure 2A**). High levels of AS Uchl1 RNA are detected in neonatal corpus striatum, cortex, hippocampus and medulla oblungata.

As each brain region is composed of several cellular types of neuronal and non-neuronal origin, we monitored S/AS expression in primary brain cells as well as in neurospheres. Again, we found that the expression of S and AS Uchl1 RNAs is coregulated. Both transcripts are highly expressed in neurons as compared to non-neuronal cells, with those from the Striatum, the Substantia Nigra and Raphe being the highest (**Figure 2B**). Interestingly, values in neurospheres prepared from cells isolated from different compartments show reduced quantities as compared to primary neurons. Non-neuronal cells have very low (Schwann cells) to undetectable (astrocytes) levels of Uchl1 S/AS pair.

Therefore, expression of S and AS Uchl1 is co-regulated in the developing and adult brain.

# Analysis of TSS Usage in Mouse Uchl1 Gene Locus

To monitor promoter usage in mouse Uchl1 locus, we visualized single TSSs using the FANTOM5 Zenbu Genome Browser, a publicly available web resource tool (http://fantom.gsc.riken. jp/zenbu/) (Severin et al., 2014). To identify promoters across the genome the FANTOM5 consortium has developed a method based on tags proximity and signal decomposition (Forrest et al., 2014). To enrich for TSSs, 3 tags/library thresholds were applied and promoter subsets were defined and classified according to their robustness. For our analysis, we interrogated all hCAGE libraries pooled together and then we focused our attention on those with stronger evidences of TSS usages, as testis, cortex and primary neurons of the Substantia Nigra. As shown in **Figure 3**, three different TSSs can be identified using decomposition-based identification method and referred to as p1, p2 and p3, from 5′ to 3′ according to the sense of transcription. These drive expression of alternative variants of AS Uchl1 RNA. TSS usage is under tissue- and cell-specific regulation, as documented by different TPM values in the selected libraries. Interestingly, expression of AS Uchl1 reference sequence is validated by TSSs in testis and Substantia Nigra neurons, but this promoter does not pass the bioinformatics cutoffs. p2 and p1 are the most used promoters,

FIGURE 2 | S/AS Uchl1 are regulated during development and enriched in neurons. (A) Heat map graphical representation of S/AS expression in neonatal and adult tissues from brain and other organs. Expression values of S/AS transcripts in cortex were set to 1. (B) Heat map of S/AS Uchl1 in brain-derived primary cells and in neurospheres. Values in cortical neurons were set to 1.

giving rise to transcript variants with a longer 5′ end. It is of note that 5′ end of AS Uchl1, as identified by RACE in murine dopaminergic MN9D cell line (Carrieri et al., 2012), is positioned upstream to p1 (**Figure 3**). Finally, an additional site of transcription initiation can be observed at the 3′ end of AS Uchl1 (p3), thus identifying a yet not-annotated variant with a shorter first exon. p3 seems to be under control of a bidirectional promoter.

The protein-coding Uchl1 mRNA starts almost exclusively from an internal portion of the 5′ UTR of the annotated transcript. It is shorter and independently validated by mm9 mRNAs (**Figure 3**). Interestingly, an alternative promoter for Uchl1 is also located around exon 3 and drives expression in testis (**Figure 3**, black arrow). This TSS identifies an annotated but yet uncharacterized transcript (AK170728), which potentially encodes for a shorter Uchl1 protein, lacking a canonical initiation methionine. Whether such protein is indeed functional remains to be established.

### Nurr1 Activity Regulates AS Uchl1 Expression

The genomic region −1500/+1000 around the annotated AS Uchl1 TSS was then scanned for the presence of Transcription Factor Binding Sites (TFBSs). A NGFI-B element was identified in position −1230/−1222 to the AS Uchl1 TSS as defined by RACE (**Figure 4A**). This TFBS is the target of the Nurr subfamily of nuclear receptors including Nurr1, a key dopaminergic transcription factor required for late-dopaminergic differentiation and crucial for the expression of several dopaminergic-specific genes like VMAT2, AADC, DAT, and TH (Castro et al., 2001).

To study the role of this transcription factor in AS Uchl1 RNA transcription, we took advantage of mouse dopaminergic MN9D cell line overexpressing Nurr1 under a doxycicline inducible promoter (iMN9D cells) (Hermanson et al., 2003). In steady-state conditions, levels of Nurr1 protein are almost undetectable and AS Uchl1 is expressed at low level. Upon drug treatment, Nurr1 expression increases and Nurr1 target genes are induced (Hermanson et al., 2003). As a consequence, differentiation of DA neurons occurs (**Figure 4B**).

We then carried out chromatin immunoprecipitation (ChIP) experiments. iMN9D cells were treated with doxocycline for 12 h to achieve high levels of Nurr1 protein. Chromatin-protein complexes were then immunoprecipitated with anti-Nurr1 or control (IgG) antibodies and bound genomic DNA was quantified by qPCR using primers for Nurr1 response elements in AS Uchl1, Osteocalcin and Vescicular Monoamine Transporter 2 gene promoters. PCR reactions generated only the expected specific amplicon, as detected by gel electrophoresis and melting curve analysis (data not shown). As shown in **Figure 4C**, Nurr1 binding was significantly enriched relative to IgG control in AS Uchl1 promoter region.

Nurr1 induction led to a rapid up-regulation of AS Uchl1 RNA levels starting from 12 h, with kinetics comparable to the one observed for VMAT2, a well-known primary target of Nurr1. Within 48 h, both AS Uchl1 and VMAT2 mRNAs reached their peak of induction, while decreasing at later time points. Under these conditions, Nur77, another member of nuclear receptors family targeting NGFI-B motif, is undetectable (**Supplementary Figure 1**).

Interestingly, we found that transcription of sense proteincoding Uchl1 mRNA was poorly linked to Nurr1 activity, as the kinetics and strength of Uchl1 mRNA up-regulation was different from that of VMAT2 and AS Uchl1 (**Figure 4B**).

# Expression of Mouse AS Uchl1 is Down-Regulated in Neurochemical Models of PD In Vitro and In Vivo

We then investigated the behavior of Uchl1 S/AS pair in neurochemical models of PD. First, we analyzed the effects of intoxication on S/AS Uchl1 levels in vitro. When iMN9D cells were exposed to 100µM 1-methyl-4-phenylpyridinium (MPP+) for 16 h, we observed that expression of both transcripts was altered and a statistically significant co-reduction could be measured. While AS Uchl1 RNA was reduced to only 25% of its physiological level, the impact on Uchl1 mRNA was less pronounced, with only 30% reduction (**Figure 5A**).

To test whether S/AS transcription is regulated during DA neuron intoxication in vivo, we injected mice with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), following a sub-acute protocol (Jackson-Lewis and Przedborski, 2007). To uncouple transcriptional control of S/AS expression from down-regulation due to cell loss, we resolved to laser capture microdissection of fluorescently labeled DA neurons from TH-GFP transgenic mice to purify equal numbers of cells from untreated and treated animals. Under these conditions, surviving DA neurons showed an almost complete loss of AS Uchl1 transcript but a modest downregulation of Uchl1 mRNA (**Figure 5B**) recapitulating what we had observed in vitro in iMN9D cells treated with MPP+.

Altogether, these data show that AS Uchl1 is a component of the Nurr1-dependent dopaminergic gene network and is down-regulated in neurotoxicity.

# Discussion

One of the key features of genomes' organization is that the large majority of genes share their genomic region with another gene on the opposite filament forming S/AS pair (Carninci et al., 2005; Derrien et al., 2012). Massive antisense transcription seems to be a common feature of cells ranging from bacteria to human (Katayama et al., 2005; Van Dijk et al., 2011). Despite some provocative examples we poorly understand how each locus decides between repressive and activating responses to antisense transcription suggesting we are still missing the basic principles of regulatory choices and molecular switches. This is particularly relevant when we consider that lncRNAs and antisense transcription may be exploited in RNA therapeutics.

The S/AS pair at the Uchl1 locus is organized according to the configuration where a protein-encoding gene presents a lncRNA on the opposite strand, overlapping "head-to-head" at their 5′ ends. AS Uchl1 is the representative member of SINEUPs, a new functional class of natural and synthetic antisense lncRNAs that activate translation. Their activity depends on the combination of two RNA elements: the overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). The ED is required for the increase of translation of the protein-coding sense mRNA and the BD is targeting the activity to the sense transcript.

UchL1 is one of the most abundant proteins in the brain where it is expressed in selected neuronal cell types. We have previously shown that in iMND9 cells, the very same cell line used in this study, its mRNA is associated to light polysomes for basal translation. In these conditions AS Uchl1 is mainly restricted to the nucleus where it exerts an unknown function. S/AS transcripts are also co-expressed in mesencephalic DA cells in vivo where they retain their differential subcellular localization. Upon inhibition of mTOR activity by rapamycin, AS Uchl1 shuttles to the citoplasm where it facilitates Uchl1 mRNA loading to heavy polisomes for a more efficient translation and increase in cellular UchL1 protein levels. Both rapamycin treatment and Uchl1 over-expression are considered neuroprotective suggesting that antisense-mediated translation may be part of a pro-survival cellular response to stress.

A first step toward a better understanding of antisense transcription consists in studying how its expression is regulated. Increasing information is accumulating over the role of DNA methylation and selected TFs such as Sox2, Oct4, and Nanog on the control of long intergenic non-coding RNAs (lincRNAs) (Guttman et al., 2009) whereas very little is known about the transcriptional regulation of the lncRNAs component of S/AS pairs. Recent genome-wide studies on human cell lines evidence epigenetic regulation of AS lncRNAs expression, with accumulation of histone marks and RNA polymerase II occupancy (Conley and Jordan, 2012). Interestingly, TSSs in antisense orientation frequently associate with transposable elements, suggesting that repetitive sequences might contribute to regulation of AS transcription (Conley et al., 2008; Kapusta et al., 2013). Few genespecific examples exist in which transcriptional control of a natural AS transcript has been investigated. This is the case, for example, of S/AS Msx1 in which the promoter region of AS RNA contains binding sites for Msx1 transcription factor, thus generating an auto-regulatory loop of S/AS regulation (Petit et al., 2009; Babajko et al., 2011). In prostate cancer cell line, the expression of CTBP1-AS is regulated by binding of androgen receptor at the 5 ′ of its TSS. Upon androgen-dependent activation of CTBP1- AS, the overlapping protein-coding gene (CTBP1) is repressed (Takayama et al., 2013).

Here we find that almost all the FANTOM5 libraries present evidences of Uchl1 transcription while AS Uchl1 is expressed only in 40% of them. This data raise the question about how UchL1 protein synthesis is regulated in physiological conditions and upon stress in the absence of antisense transcription. It will be interesting to assess whether Uchl1 mRNA is normally associated to heavy polisomes and whether mTOR inhibition or stress are able to increase UchL1 protein levels according to an antisense–independent mechanism. This is particularly relevant considering that UchL1 is target of oxidative stress inactivation and is down-regulated in post-mortem brains of neurodegenerative diseases. In summary these data may raise the interesting possibility of differences in the anti-stress control and function of Uchl1 expression dependent on the presence of AS Uchl1 RNA.

We did not find any example of AS Uchl1 expression in the absence of Uchl1. Therefore, it is not surprising that when both S/AS pair transcripts are present they show a concordant pattern of expression. The highest levels of AS Uchl1 have been found in Substantia Nigra, striatum and diencephalon. This analysis confirms AS Uchl1 is present in selected regions of the brain as shown in Carrieri et al. (2012).

Interestingly, neonatal neurons express larger quantity of AS Uchl1 RNA than adult tissues. This is in line with recent genome-wide and gene-specific studies that indicate spatiotemporal regulation of AS lncRNAs expression during cerebral corticogenesis (Ling et al., 2011; Lipovich et al., 2013) and aging (Pardo et al., 2013) (Francescatto et al., FANTOM5 satellite, submitted).

Even when AS Uchl1 is highly expressed, its levels are lower than Uchl1 mRNA. However, we know that AS Uchl1 is able to increase the amount of UchL1 protein through its activity on Uchl1 mRNA. The quantity of UchL1 protein in vivo is the sum of several molecular events ranging from transcription to mRNA localization, rate of protein synthesis and degradation. It is unclear what is the life cycle of a single Uchl1 mRNA from transcription to its association to ribosomes or stress granules and P bodies. Therefore, it remains crucial to determine which fraction of total cellular Uchl1 mRNA is associated to light polisomes in physiological conditions and how much is available for RNA-RNA interaction and association to heavy polisomes in an antisense-dependent fashion under stress.

By decomposition-based identification method three different promoters can be defined while evidences for additional TSSs are shown. While p1 and p2 extend the overlapping region at genomic level respect to the annotated sequence of reference, a previously reported cDNA clone obtained with RACE starts 5′ to p1. p3 identifies a non-overlapping transcript probably involved in a bidirectional promoter. Interestingly, a potential internal alternative TSS for Uchl1 is evidenced in testis and Substantia Nigra suggesting the possibility of a N-terminal truncated protein.

At 1230 nt from the TSS of AS Uchl1 identified with RACE, a DNA binding site for Nurr1 has been bioinformatically discovered and experimentally validated. Nurr1 is a member of the nuclear receptor superfamily, whose expression is crucial for DA neurons differentiation and maintenance in the adult (Zetterstrom et al., 1997; Saucedo-Cardenas et al., 1998; Kadkhodaei et al., 2009, 2013). Importantly, reduced quantities of Nurr1 in adult mice increase DA cells vulnerability to neurotoxic insults (Le et al., 1999).  In summary, Nurr1 controls the major transcriptional axis for differentiation of DA neurons and maintenance of their integrity.

This work strongly suggests that AS Uchl1 is a component of Nurr1-dependent transcriptional network. This functional interaction adds a new layer to the complexity of circuitry that may contribute to DA cells' homeostasis.

We then prove that neurochemical intoxication of DA cells down-regulates S/AS expression in vitro and in vivo with a kinetics that impact more strongly AS Uchl1 than its overlapping sense mRNA. This is different from the consequences of pharmacological inhibition of mTOR where no effects on transcription were observed (Carrieri et al., 2012). Further studies are needed to understand whether survival or apoptotic outocomes of different treatments reside, at least in part, on the ability to regulate antisense RNAs at transcriptional or post-transcriptional levels.

In summary, antisense transcription imposes an additional level of regulatory networks to control the activity of proteincoding genes. Determining the repertory of transcription factors controlling the expression of AS RNAs under physiological and pathological conditions will further contribute to our understanding on the biological functions of AS lncRNAs in health and disease. Finally, since increasing evidences suggest Uchl1 over-expression could be beneficial in neurodegenerative diseases, the use of AS Uchl1 as RNA-based drug may represent a new therapeutic strategy.

# Acknowledgments

We are indebted to all the members of the SG lab for thoughtprovoking discussions and to Cristina Leonesi for technical support. We are grateful to SISSA technical and administrative staff, especially to J. Franzot, T. Bigiarini, H. Krmac, Monica Sirk, Simonetta Vetter, and Erica Maran. This work was supported by the Italian Ministry of Education, University and Research (FIRB grant prot. RBAP11FRE9) to SG and FP by the Research Grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies and by the "Program for Development of Basic Technology to Discover Innovative Biotechnology-Based Drugs" from The Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to PC.

# Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fncel.2015. 00114/abstract

# References

Supplementary Figure 1 | Nur77 expression in iMN9D cells. iMN9D cells were treated with doxycyclin for 48 h. Expression of Nur77 was monitored by qRT-PCR with specific primers. Expression of VMAT2 mRNA was used as positive control for Nurr1-mediated activation. Data were analyzed using the 2 −1Ct method. Data indicate mean ± SD. Nurr1 expression was verified by western blotting.

disease and drives rapid feed-forward regulation of beta-secretase. Nat. Med. 14, 723–730. doi: 10.1038/nm1784

**Conflict of Interest Statement:** SG, PC, CC, AF, and SZ are named inventors in patent issued in the US Patent and Trademark Office on SINEUPs and licensed to TransSINE Technologies. SG and PC are co-founders of PARKscreen, an Italian SME aimed to use and develop therapeutic SINEUPs.

Copyright © 2015 Carrieri, Forrest, Santoro, Persichetti, Carninci, Zucchelli and Gustincich. 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.

# SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells

Silvia Zucchelli 1, 2 † , Francesca Fasolo1 †, Roberta Russo1 , Laura Cimatti 1 , Laura Patrucco2 , Hazuki Takahashi 3 , Michael H. Jones 4 , Claudio Santoro2 , Daniele Sblattero2 , Diego Cotella2 , Francesca Persichetti 2 , Piero Carninci 3 and Stefano Gustincich1 \*

*1 Scuola Internazionale Superiore di Studi Avanzati, Area of Neuroscience, Trieste, Italy, 2 Dipartimento di Scienze della Salute, Universita' del Piemonte Orientale, Novara, Italy, 3 Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan, 4 Cell Guidance Systems, Cambridge, UK*

#### Edited by:

*Rosanna Parlato, Ulm University, Germany*

#### Reviewed by:

*Mohammadreza Hajjari, Shahid Chamran University of Ahvaz, Iran Rohit Mathur, M.D. Anderson Cancer Center, USA*

#### \*Correspondence:

*Stefano Gustincich, Scuola Internazionale Superiore di Studi Avanzati, Sector of Neurobiology, Via Bonomea 265, 34136 Trieste, Italy<EMAIL_ADDRESS>
† *These authors have contributed equally to this work.*

Received: *10 December 2014* Accepted: *20 April 2015* Published: *13 May 2015*

#### Citation:

*Zucchelli S, Fasolo F, Russo R, Cimatti L, Patrucco L, Takahashi H, Jones MH, Santoro C, Sblattero D, Cotella D, Persichetti F, Carninci P and Gustincich S (2015) SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells. Front. Cell. Neurosci. 9:174. doi: 10.3389/fncel.2015.00174* Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Keywords: SINEUP, long non-coding RNA, antisense, protein expression, cell lines

# Introduction

Large genomic projects such as ENCODE (Djebali et al., 2012) and FANTOM (Forrest et al., 2014) have shown that the majority of the mammalian genome is transcribed, thus generating a previously underestimated complexity in gene regulatory networks. Protein encoding genes present a large repertory of alternative Transcription Start Sites (TSSs) that may drive transcription in a cell type-specific manner (Valen et al., 2009). Different 5′UTRs may contain information for mRNA sorting to neuronal compartments as well as for stimulus-dependent translation. Furthermore, in addition to 25000 genes encoding for proteins, at least an equal number of long non-coding RNA (lncRNA) genes have been identified so far. These generate >200 base pairs long transcripts that do not encode for proteins. About one third of annotated lncRNAs overlaps with proteincoding genes (Derrien et al., 2012). Many of these are transcribed from the opposite strand forming sense/antisense (S/AS) pairs (Katayama et al., 2005; Derrien et al., 2012).

The nervous system appears as a privileged site for lncRNA expression, as the vast majority of these transcripts is brainenriched and regulates neuronal development and functions (Qureshi and Mehler, 2012). Furthermore, a complex network of natural S/AS pairs may participate in brain development and homeostasis in physiological conditions. Interestingly, an increasing number of lncRNAs are associated with brain dysfunction and extensive AS transcription has been measured in loci associated to hereditary neurodegenerative diseases (Zucchelli et al., submitted).

Manipulating RNA expression in vivo has been proposed as new strategy for molecular therapy. Special attention has been devoted to small antisense oligonucleotides (ASOs) and siRNAs as tools to decrease gene expression of pathological target genes such as, for example, mutant huntingtin in Huntington's disease (Kordasiewicz et al., 2012; Yu et al., 2012).

An equally challenging large group of diseases would strongly benefit from the discovery of RNAs that can increase protein levels of genes for which low expression is pathogenic. Several hereditary intellectual and cognitive disabilities are haploinsufficiencies where only a single functional copy of a gene is unable to produce sufficient protein to maintain a physiological condition (Van Bokhoven, 2011). Therefore an RNA-based drug that can restore physiological amounts of the target protein can in principle be curative. Unfortunately, no molecules that can increase protein levels of a specific mRNA type in vivo have been found to date.

In search for AS transcripts that may regulate the expression of Parkinson's disease (PD)-associated genes, we recently discovered AS Uchl1, a natural lncRNA antisense to Ubiquitin carboxyl-terminal esterase L1 (Uchl1/PARK5) (Carrieri et al., 2012). AS Uchl1 is a nuclear-enriched transcript, that is expressed in dopaminergic neurons in the Substantia Nigra, the target of PD neurodegeneration, and is down-regulated upon PDmimicking intoxication in vitro and in vivo (Carrieri et al., 2015). AS Uchl1 activity increases UchL1 protein synthesis at the post-transcriptional level. Upon stressful insults, AS Uchl1 shuttles from the nucleus to the cytoplasm, where it induces Uchl1 mRNA association to heavy polysomes to increase its translation (Carrieri et al., 2012). AS Uchl1 activity depends on two functional domains: the overlapping region that defines target specificity and the inverted SINE element of B2 subclass (invSINEB2) that confers protein synthesis activation. This functional organization is shared with other lncRNAs part of S/AS pairs in the mouse genome. Finally, by substituting the overlapping region with a sequence antisense to the Green Fluorescent Protein (GFP) mRNA, this synthetic RNA was able to increase GFP protein synthesis with a post-transcriptional mechanism (Carrieri et al., 2012).

The potential scalability of AS Uchl1-derived synthetic lncRNAs to a platform of mRNA-specific translation enhancers remained to be addressed.

Here we propose that AS Uchl1 is the representative member of a new functional class of natural and synthetic RNAs that increase protein synthesis. We name these RNAs as SINEUPs for their activity requires an invSINEB2 element (SINE) to UPregulate translation of partially overlapping sense mRNAs. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest.

This work shows that synthetic SINEUPs are a versatile tool to increase synthesis of target proteins of interest paving the way for future applications of SINEUPs as molecular biology reagents and for manufacturing of recombinant proteins. Most importantly, SINEUPs may be directed to selected mRNA species in vivo representing a new type of RNA-based drug for molecular therapy.

# Materials and Methods

# Constructs

Plasmids expressing target proteins were previously described. In particular, for this study we used pEGFP-C2 (Carrieri et al., 2012), pcDNA3-2XFLAG-DJ-1 (Herrera et al., 2007; Zucchelli et al., 2009), pcDNA3-2XFLAG-TTRAP (Zucchelli et al., 2009; Vilotti et al., 2012), pcDNA3-2XFLAG-Hba (Biagioli et al., 2009) (Codrich et al., manuscript in preparation) and pcDNA3- 2XFLAG-TRAF6 (Zucchelli et al., 2010, 2011).

Target specific SINEUPs were constructed using pcDNA3- 15 ′ -ASUchl1 as backbone (Carrieri et al., 2012). SINEUPbackbone lacks the region of overlap (BD) to Uchl1 and retains AS Uchl1 ED with inverted SINEB2, Alu sequence and 3′ tail. SINEUP target-specific BDs were designed, in antisense orientation, around the ATG of protein-coding sequence with a −40/+32 anatomy.

SINEUP targeting EGFP (AS-GFP, here named SINEUP-GFP) has been described in Carrieri et al. (2012). SINEUP targeting FLAG-tagged proteins (SINEUP-FLAG) was cloned with the following primers (5′ to 3′ orientation):

**Abbreviations:** lncRNA, long non-coding RNA; AS, antisense; SINE, short interspersed nuclear element; invSINEB2, inverted SINE of B2 subfamily; PD, Parkinson's disease; TRAF6, tumor necrosis factor receptor associated factor 6; TTRAP/TDP2, TRAF and TNF receptor associated protein/tyrosyl-DNA phosphodiesterase 2; Hba, hemoglobin alpha chain.

### FWD AS 2xFLAG:

ATATCTCGAGAATTCCTTGTCATCGTCGTCCTTGTAGT CCATCAATTCCAGCACACTGGCGGCCGT

REV AS 2xFLAG:

GAGAGATATCCTCGGATCCACTAGTAACGGCCGCCAG TGTGCTGGAATTGATGGACTACAAGGACG

Primers were annealed, elongated by PCR, digested and ligated into XhoI-EcoRV sites of SINEUP-backbone.

Short SINEUP targeting GFP (miniSINEUP-GFP) was generated combining BD of SINEUP-GFP and ED of AS Uchl1. Briefly, inverted SINEB2 was PCR amplified and cloned into EcoRI and HindIII sites of pcDNA3.1(-). SINEUP-GFP BD was subsequently added to the inverted SINEB2-containing plasmid at XhoI and EcoRI sites to obtain miniSINEUP-GFP. The following primers were used:

FWD EcoRI InvSINEB2: TATAGAATTCCAGTGCTAGA GGAGG

REV HindIII InvSINEB2: GAGAAAGCTTAAGAGACTG GAGC

FWD ApaI all O/L: TATAGGGCCCTCTAGACTCGAG

REV EcoRI O/L GFP20: GAGAGAATTCCAGCACAG TGGCGGCCGC

SINEUPs targeting DJ-1 were generated by annealing and PCR-based method (−40/+32) or with annealing and ligation of phosphorylated oligonucleotides (−40/+4), using the following primers:

SINEUP-DJ-1 (−40/+32) FWD:

ATATCTCGAGGCCAGGATGACCAGAGCTCTTTTGG

AAGCCATTTTTATGTTATATGTTT

SINEUP-DJ-1 (−40/+32) REV:

GAGAGATATCTTTCAGCCTGGTGTGGGGCTTGT

AAACATATAACATAAAAATGGCTT

SINEUP-DJ-1 (−40/+4) FWD:

TCGAGCCATTTTTATGTTATATGTTTACAAGCCCCACA

CCAGGCTGAAA SINEUP-DJ-1 (−40/+4) REV:

TTTCAGCCTGGTGTGGGGCTTGTAAACATATAACAT

AAAAATGGC

All constructs were verified by sequencing.

# Cell Lines and Transfection

HEK 293T/17 cells were obtained from ATCC (Cat. No. ATCC-CRL-11268 293T/17) and maintained in culture with Dulbecco's Modified Eagle Medium (GIBCO) supplemented with 10% FBS (SIGMA) and 1% antibiotics (penicillin/streptomycin), as suggested by the vendor. HepG2 cells were kindly provided by Professor Collavin L. from the University of Trieste, Italy (Lunardi et al., 2009). HepG2 and SK-N-SH were cultured in Eagle's minimal essential medium (SIGMA) supplemented with 10% FBS, 1% antibiotics, 1% GlutaMAX and 1% nonessential aminoacids. HeLa cells were grown with DMEM supplemented with 10% FBS and 1% antibiotics as previously described (Angelini et al., 2007). SH-SY5Y cells were maintained in culture as previously described (Zucchelli et al., 2009). BE(2)M17 were grown in 1:1 MEM-Glutamax (GIBCO)/F12 (GIBCO) supplemented with 10% FBS, 1% antibiotics, and 1% non-essential aminoacids.

When required, HEK 293T/17 cells were treated with rapamycin (SIGMA) at 1µM for 1 h or at 100 nM for 16 h. DNA damage was induced by doxorubicin (SIGMA), at 1µM for 1 h or 500 nM for 16 h.

HEK 293T/17 cells were transfected with Fugene HD (Roche) and Lipofectamine 2000 (Life Technologies), following manufacture's instruction. HepG2, HeLa, SH-SY5Y, BE(2)M17 and SK-N-SH cells were transfected with Lipofectamine 2000. All cells were transfected with a 1:6 ratio between sense and SINEUP encoding plasmids, maintaining the conditions described for S/AS Uchl1 (Carrieri et al., 2012). In detail, cells were plated in 6-well plates the day before transfection at 60% (for Fugene HD protocol) or 80–90% (for Lipofectamine protocol) confluency. For transfection with Fugene 0.3µg pEGFP and 1.8µg SINEUP plasmid were used; for Lipofectamine 2000 0.6µg pEGFP and 3.4µg SINEUP. Cells were collected at 24 h (HeLa cells) or 48 h (HEK 293T/17 and HepG2 cells) after transfection and split in two samples for RNA extraction and Western Blot analysis.

For SINEUPs targeting endogenous mRNAs, SINEUPencoding plasmid was transfected at the highest dose (4 µg) following manufacture's instructions.

Data of RNA and protein levels were obtained from the same transfection in each replica.

# Western Blot

For Western blot analysis, cell pellets were directly resuspended in Laemli sample buffer, briefly sonicated, boiled and loaded on poly-acrilamide gels.

Primary antibodies used in this study include anti-GFP rabbit polyclonal antibody (Life Techologies, Cat. No. A6445), used 1:1000, anti-FLAG M2 (SIGMA, Cat. No. 3165), 1:1000, anti-βactin (SIGMA), 1:5000, and anti-TRAF6 (Abnova), used 1:500. To detect endogenous DJ-1 protein an antibody produced in our laboratory was used (Zucchelli et al., 2009; Foti et al., 2010). For the detection, anti-mouse-HRP or anti-rabbit-HRP (Dako) in combination with ECL (GE Healthcare) was used. Image detection was performed with Alliance LD2-77WL system (Uvitec, Cambridge). Image quantification was done using Adobe Photoshop CS5.

# RNA Isolation, Reverse Transcription and Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from cells using RNeasy Mini Kit (QIAGEN) following manufacturer's instructions. RNA was treated with on-column DNase I (QIAGEN) followed by a second DNase I digestion in solution (Ambion). Two rounds of DNase digestion were required to avoid plasmid DNA contamination in this experimental setting. Single strand cDNA was prepared from 1µg of purified RNA using the iSCRIPT™ cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. qRT-PCR reaction was performed on diluted cDNA (1:20) using SYBR-Green PCR Master Mix (Applied Biosystem) and an iCycler IQ Real time PCR System (Bio-Rad). Relative expression was calculated with the 11Ct method (Schmittgen and Livak, 2008). Oligonucleotide sequences of primers used in this study for GFP and GAPDH (Carrieri et al., 2012), DJ-1 (Foti et al., 2010), TTRAP (Zucchelli et al., 2009), TRAF6 (Zucchelli et al., 2010) and Hba (Biagioli et al., 2009) were previously described.

SINEUP-GFP, SINEUP-FLAG and SINEUP-DJ-1 were detected with primers designed on the 3′ end of AS-Uchl1 (mAS Uchl1 FWD and REV, Primers 3′ ) (Carrieri et al., 2012). MiniSINEUP-GFP was quantified using the following primers: pTSinvB2 FWD-RT (CAGTGCTAGAGGAGGTCAGAAGA) and pTSinvB2 REV-RT (GGAGCTAAAGAGATGGCTCAG CACTT).

# Cellular Fractionation

For fractionation experiments, GFP/SINEUP-GFP were transfected in 10 cm plates at 1:6 ratio using Lipofectamine 2000. Nucleo cytoplasmic fractionation was performed as previously described (Wang et al., 2006). Nucleus and cytoplasmic RNAs were extracted using Trizol reagent (Invitrogen) following manufacturer's instruction. RNA was eluted and treated with DNase I. The purity of the cytoplasmic fractions was confirmed by qRT-PCR on pre-ribosomal RNA using the following primers (Murayama et al., 2008):

FWD 5′ -GAACGGTGGTGTGTCGTTC-3′ REV 5′ -GCGTCTCGTCTCGTCTCACT-3′

# Statistical Analysis

All data are expressed as mean ± standard deviation on n ≥ 3 replicas. Statistical analysis was performed using Excel software. Statistically significant differences were assessed by Student's ttest. Differences with p < 0.05 were considered significant.

# Results

# SINEUPs: Definition and Design

As shown in Carrieri et al. (2012), AS Uchl1 stimulates translation of partially overlapping sense protein-coding mRNAs with no effects on RNA levels.

Here we propose AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named these lncRNAs as SINEUPs since they take advantage of an embedded invSINEB2 element to UP-regulate translation. Therefore SINEUPs can be considered the first example of gene-specific inducers of protein synthesis.

SINEUPs display a modular architecture (**Figure 1A**). In the 5′ region, SINEUPs contain the sequence that overlaps, in antisense orientation, to the sense protein-coding mRNA. We named this sequence as SINEUP's Binding Domain (BD) since it provides target selection and SINEUP specificity by RNA-RNA base pairing. In AS Uchl1, BD is 73 base pair long, centered across the ATG with a −40/+32 configuration, spanning part of Uchl1 5′UTR and a portion of its CDS (Carrieri et al., 2012). The remaining SINEUP sequence presents the embedded invSINEB2 element in the non-overlapping part of the transcript. Since this region has been proven essential for protein synthesis upregulation, we defined it as Effector Domain (ED) (**Figure 1A**).

In Carrieri et al., we showed that this modular structure is shared with several natural lncRNAs component of S/AS pair in the mouse genome. For AS Uxt we proved it is able to increase UXT protein synthesis with no effects on Uxt mRNA levels. Therefore these define the first entries in the list of natural SINEUPs.

Given their modular structure, target-specific synthetic SINEUPs can be designed at will by manipulating AS Uchl1 sequence (**Figure 1B**). In Carrieri et al., we have designed the first synthetic SINEUP directing its activity to GFP mRNA by swapping BDs. However, any invSINEB2 sequence from other natural SINEUPs can in principle sustain activity and be considered potential ED.

Provided with the exact TSS for the target gene, sequence of interest is extrapolated centered across the initiating ATG. After reverse-complement manipulation, gene-specific BD is generated by annealing and PCR amplification of specific oligonucleotides. Target-specific SINEUP is then obtained by cloning specific BDs upstream to the SINEUP effector domain. For expression in mammalian cells, SINEUPs are cloned into pcDNA3.1 plasmid. Vectors for retroviral and lentiviral packaging can also be efficiently used (data not shown).

# SINEUPs Work in vitro in Different Cell Lines

Mammalian cell cultures in vitro are routinely used as model systems to study the molecular mechanisms of gene functions as well as cell factories to produce therapeutic proteins. Considering the flexibility of SINEUP technology and its potential applications in molecular biology experiments, protein manufacturing and therapeutics, we investigated the efficacy and reproducibility of SINEUPs in different cell lines in vitro. To this purpose we selected hepatocellular carcinoma HepG2 cells, for their use as a model system by large multicenter consortia, epithelial carcinoma HeLa cells for their wide use in cell biology and in therapeutic protein production as well as HEK 293T/17 cells as positive control of SINEUP activity. As a representative synthetic SINEUP, we took advantage of SINEUP-GFP to increase GFP protein levels in transient overexpression experiments.

We estimated SINEUP activity as fold changes in protein levels encoded by targeted mRNAs in the presence/absence of SINEUP with mRNA amounts kept constant (p > 0.05).

Similar fold-changes were observed for SINEUP activity with GFP target at 24 and 48 h after transfection (data not shown). However timing for optimal activity was cell-line dependent, as best conditions were found at 24 h in HeLa and 48 h in HEK 293T/17 and HepG2 cells (**Figures 2A–C**).

In HEK 293T/17 cells we obtained an average 2.4 fold change (**Figure 2A**), confirming previously published data (Carrieri et al., 2012) on an independent batch of cells and on a larger cohort of transfections. SINEUP activity ranged from a minimum of 60% induction to a maximum of 400% (**Figure 2D**). SINEUP effect in transfected HEK 293T/17 cells was not enhanced upon stressful stimuli such as rapamycin and DNA-damage inducing drug doxorubicin (**Supplementary Figure 1**). HepG2 cells and HeLa cells proved to support SINEUP activity (**Figures 2B,C**), with an average induction of 1.65 and 1.82-fold, respectively. Minimal values were 20% in HepG2 and 40% in HeLa cells, and top effect was 250 and 220% (**Figure 2D**). No statistical differences could be measured in SINEUP activity between the three cell lines (p > 0.05), albeit HEK 293T/17 cells tended to be more effective (**Figure 2D**).

We observed that SINEUP activity is maintained independently of the reagent used for transfection. In HEK 293T/17 cells an average of 2.4 fold change could be measured with Lipofectamine (n = 5 experiments) and 2.3 with Fugene HD (n = 6 experiments) (data not shown). Under these experimental conditions, RNA from transfected SINEUP-GFP was detected in the cytoplasmic fraction although a prominent accumulation in the nucleus was evident (**Supplementary Figure 2**). Interestingly, sense GFP mRNA was equally distributed between cytoplasmic and nuclear compartments. Altogether these data indicate that SINEUPs can be used in vitro in different cell systems to up-regulate proteins of interest.

# SINEUPs can be Designed to Increase Production of Target Proteins of Interest

The modular structure of SINEUPs predicts that by swapping the BD with an appropriate sequence it should be possible to redirect SINEUP activity to target mRNA of interest.

To test the flexibility of BD design, we generated a SINEUP molecule targeting the commonly used FLAG tag sequence. FLAG-specific SINEUP would be able to act at a post-transcriptional level increasing the quantities of proteins expressed in frame with an N-terminal FLAG tag. SINEUP targeting FLAG-tagged proteins (SINEUP-FLAG) was designed to mimic the molecular anatomy of SINEUP-GFP. In particular, SINEUP-FLAG BD encompasses −40 nucleotides (in pcDNA3 plasmid backbone) before FLAG-initiating Met and +32 bases covering the first FLAG tag sequence (**Figure 3A**).

We took advantage of a series of protein-coding genes available in the laboratory to test SINEUP-FLAG activity.  HEK 293T/17 cells were transfected with plasmids for FLAG-tagged targets in combination with SINEUP-FLAG (+SINEUP). Cells transfected with an empty vector were used as control (-SINEUP). SINEUP activity was measured quantifying protein levels by Western blot and RNA amounts by qRT-PCR. We found that the quantity of three of four FLAG-tagged proteins that we tested was modulated by coexpression of SINEUP-FLAG (**Figure 3B**). SINEUP effect was different toward the three targets, ranging from 1.5 to 3.0 fold changes. FLAG-TRAF6 showed the strongest SINEUPmediated activation with a protein induction consistently in the range of 2.6 to 3.0 fold (**Figure 3B** and data not shown). No effect on TRAF6 mRNA was present, as expected (p = 0.46). Interestingly, increased TRAF6 levels could

be measured when probing lysates with anti-TRAF6 specific antibody (**Supplementary Figure 3**).

antibody. β-actin was used as loading control. Fold-induction was calculated

A 50% up-regulation was measured with FLAG-DJ-1 and 90% with FLAG-Hba. In both cases, sense mRNA in -SINEUP and +SINEUP transfections was not statistically different (p = 0.36 in FLAG-DJ-1 samples and p = 0.38 in FLAG-Hba).

A modest activation was observed with FLAG-TTRAP where a 10–20% increase was typically observed by Western blot. However, this was consistently accompanied by a similar modulation of TTRAP mRNA levels (**Supplementary Figure 4**), thus excluding this effect from SINEUP definition.

In summary synthetic SINEUPs can be designed to commonly used tag sequences and the same SINEUP can dictate translation of different tagged proteins of interest.

# MiniSINEUPs Containing Exclusively BD and ED are Active

statistical difference was present between the three cell lines (*p* > 0.05).

A major limitation in the use of naked RNA for in vitro and in vivo applications is the instability and low cellular permeability of long molecules. Chemical modifications can bypass such limits, but with specific constraints in RNA length. Synthetic SINEUPs derived from natural AS Uchl1 are about 1200 nucleotides (nt) long with BD of 72 base pairs, ED of 170 base pairs in addition to intervening sequences, a partial Alu element (73 base pairs) and a 3′ tail. This length is suitable for delivery systems such as viral vectors, but incompatible with the use of SINEUPs as naked RNA therapeutic molecules. Therefore, we aimed at synthesizing the shortest functional SINEUP that retains its translation enhancement activity. MiniSINEUP-GFP

293T/17 cells transfected with pcDNA3-2XFLAG-TRAF6, DJ-1 and Hba, as indicated. Protein (top) and RNA (bottom) levels were analyzed by

quantities were stable (*p* > 0.05). Data indicate mean ± standard deviation and are representative of three independent experiments.

was obtained combining SINEUP-GFP BD and ED from AS Uchl1 (**Figure 4A**) giving rise to a ≈250 nt long transcript. When transfected in HEK 293T/17 cells miniSINEUP-GFP promoted a 2.5 fold increase in GFP protein levels with unaffected mRNA quantities (p = 0.11) (**Figure 4B**). Interestingly, the activity of miniSINEUP was comparable to that obtained with SINEUP-GFP (1.6 fold in this experiment, 2.4 average increase in HEK 293T/17 cells, **Figure 2D**). Under these conditions, we observed a 10-fold excess of miniSINEUP-GFP RNA relative to canonical SINEUP-GFP, as expected from its reduced size. Despite elevated RNA quantities, no impact was observed on GFP mRNA, proving that miniSINEUP retains the very same post-transcriptional mechanism of its full-length counterpart.

# SINEUPs can be Targeted to Endogenous mRNAs of Interest

The use of SINEUPs as a versatile tool to increase protein synthesis is strictly dependent on their ability to act on endogenous, cellular mRNAs transcribed from a genomic locus that does not present a natural SINEUP antisense gene.

To prove this crucial point we designed synthetic SINEUPs targeted to endogenous DJ-1 mRNA, a gene involved in recessive familial Parkinson's Disease (PD). We generated two SINEUP-DJ-1 constructs with two different BD elements: −40/+32 (long, L), from −40 nucleotides (in annotated 5′ untranslated region) before DJ-1 translation start site to +32 bases in the coding sequence, as well as −40/+4 (short, S), ending at the

first nucleotide at 3′ of ATG (**Figure 5A**). Together with HEK 293T/17, we also carried out experiments in three human neuronal cell lines [SH-SY5Y, BE(2)-M17 and SK-N-SH cells] as representative of those frequently used as in vitro system to study the function of PD-associated genes. Cells transfected with an empty vector were used as control (-SINEUP).

As shown in **Figure 5B**, SINEUP activity on endogenous DJ-1 ranged from 1.7 to 2 fold with no changes in endogenous DJ-1 mRNA levels. SINEUP-DJ-1 activity was confirmed in all three human neuronal cell lines, proving the versatility of the technology (**Figures 5C–E**). Interestingly, the highest induction of DJ-1 protein levels was measured in SK-N-SH cells (3 fold).

In summary synthetic SINEUPs can be designed to mRNAs of interest that do not present a natural SINEUP in their genomic locus opening up the scalable use of SINEUPs to target endogenous protein coding transcripts of mammalian cells.

# Discussion

To our knowledge SINEUPs are the only tool available so far that uses lncRNAs to enhance translation of target proteins. Their modular structure allows synthetic design of an overlapping region (BD) to target proteins of interest, without changing the overall structure of the original lncRNA. Indeed, by extracting solely the target-selecting BD and the ED from the original lncRNA, SINEUPs can be resized to miniSINEUPs retaining their activity.

Over competing technologies, SINEUPs have two major advantages: (1) they modulate translation of target mRNAs without introducing stable genomic changes into target cells; (2) their induction of selected protein is typically in a more physiological range (2-fold) than most conventional gene replacement strategies. These features render SINEUPs (and miniSINEUPs) a potentially interesting tool for a number of applications. First, SINEUPs may be used as reagents for molecular biology. As siRNAs have become an invaluable instrument to inhibit gene expression, many cases exist in which increasing the amount of a specific protein is required. SINEUPs may be designed to a single gene of interest or to a tag that is common to several targets and achieve translation enhancement, thus formally becoming the opposite counterpart of siRNAs. This is especially relevant in the nervous system where regulation of translation has an enormous impact on synaptic plasticity and memory formation. The synthesis of specific proteins requires a fine tuned control at single synapse and spines involving the translation of selective subtypes of mRNAs. The special challenges posed by the anatomo-functional organization of the brain require cellular machinery for controlling mRNAs localization according to the morphology and connectivity of single neuronal cell types. This is achieved at least in part by the use of different TSSs leading to the synthesis of mRNA isoforms with specific 5 ′UTR containing information for subcellular localization and translation (Baj et al., 2011). SINEUPs can thus induce translation from the mRNA isoform expressed in a defined

cellular compartment of a selected neuronal cell type increasing specificity.

Here we show that a synthetic SINEUP against the endogenous mRNA for DJ-1, a gene involved in familial PD, is able to increase its protein synthesis. This is important since, to our knowledge, the mammalian genomic DJ-1 locus does not present a natural SINEUP. This experiment thus proves SINEUPs can potentially act on protein coding transcripts of mammalian cells whether or not they are under an endogenous SINEUP-mediated translational control.

Second, considering their effect on translation, SINEUPs may find applications in protein manufacturing. More than 130 therapeutic proteins are currently in use and many more are under development, including antibodies (Leader et al., 2008). Most production strategies have concentrated efforts in optimizing culture conditions and transcription of recombinant genes leaving room for improvement at post-transcriptional level. Recently, large-scale manufacturing platforms have been developed using transiently transfected cells, mainly CHO and HEK293 (Bandaranayake and Almo, 2014). The data presented here and elsewhere (Cotella et al., submitted) support the feasibility of SINEUPs and their potential to be integrated in existing platforms.

Several aspects regulating SINEUP efficacy with selected targets have still to be elucidated. Here we found that, despite identical sequence in the overlapping region, SINEUP-FLAG failed to up-regulate FLAG-TTRAP, whilst being the most effective with FLAG-TRAF6. Furthermore, the very same BD directed to a single mRNA species can increase protein levels with different efficacies according to the host cellular type. Finally, BD of different lengths can act unlike. In this context the role of the secondary structure of the target mRNAs around AUG remains unclear, although different levels of protein increase may be also accounted by turnover rates specific for each protein or cellular context. While testing synthetic SINEUPs against a large repertory of endogenous mRNAs, we have found that a major cause of apparent lack of activity is due to the wrongful assumption that cells express the Refseq mRNA isoform of the gene. As shown by genome-wide analysis of TSS usage in mammalian cells, the complexity of alternative 5′ ends of mRNAs is staggering. Therefore, when available, we routinely interrogate the FANTOM5 dataset of CAGE libraries (Forrest et al., 2014) of the very same cells used in the experiments taking advantage of the online tool Zenbu (http://fantom.gsc.riken.jp/ 5/) to identify the correct AUG-surrounding region of the mRNA of interest. Importantly, we observed that high number of cell passages negatively influence SINEUP activity (data not shown). Global CAP-dependent translation is maintained through mTOR activity (Laplante and Sabatini, 2012) and is reduced in the majority of stress conditions (Holcik and Sonenberg, 2005; Sonenberg and Hinnebusch, 2009). We have previously showed that inhibition of mTOR with rapamycin induced an increase in UchL1 protein level dependent on the activity of the natural SINEUP AS Uchl1 (Carrieri et al., 2012). This occurs by triggering shuttling of AS Uchl1 RNA from the nucleus to the cytoplasm and the consequential increased association of Uchl1 mRNA to heavy polysomes for efficient translation. Here we showed that rapamycin and other stressors do not influence the amount of protein increase triggered by synthetic SINEUPs. We may hypothesize that by overexpression we saturate the cytoplasmic content of SINEUP RNA and/or the quantity of target mRNAs that can be associated to heavy polysomes. Further dissection of the cellular pathways that control SINEUP function and identification of SINEUP-binding proteins will provide fundamental insights to improve experimental design and answer these fundamental biological questions.

Finally, lncRNAs represent a new frontier in drugdevelopment. The field of RNA therapeutics has emerged for its great potentials and is set to increase the number of targets beyond initial expectations (Kole et al., 2012). RNA-based drugs have been developed in the past two decades often relying on short non-coding molecules, ASOs or siRNAs, to degrade mRNA or miRNAs of interest. Most recently, lncRNAs appear as therapeutic targets of ASO technology (Modarresi et al., 2012), although the field is still at an early phase. The use of lncRNAs as tools to modulate gene expression is vastly unexplored. This suffers from the limited knowledge of lncRNAs' structure/function relationship and from major obstacles in delivering long RNA molecules. In this context, miniaturization of lncRNAs represents a prerequisite toward applicability in therapeutics. Here we demonstrate that SINEUPs' modular architecture can be employed to construct a miniSINEUP that maintains full-length activity with a length within the small RNA range. Knowing the ED tridimensional structure may provide further insights to help SINEUPs design and optimize activity with minimal length requirements.

In current medical practice there are several unmet therapeutic needs for increasing protein levels in vivo. Among them, haploinsufficiency is a condition that arises when the normal phenotype requires the protein product of both alleles, and reduction to 50% or less of gene function results in an abnormal phenotype. This is the cause of a wide spectrum of diseases including ataxias and intellectual and cognitive disabilities. An efficient SINEUP activity specific for the gene of interest would be in principle curative. Furthermore, in many complex and metabolic diseases the increase of pro-survival factors and dysregulated enzymes may impact the well being of patients. As an example, augmented production of neurotrophic factors has been proposed as therapeutic treatments for the majority of neurodegenerative diseases. Therefore SINEUP molecules specific for the transcripts selectively expressed at the site of injury may potentially slow or stop disease progression. Furthermore, it may avoid the unwanted side effects of unregulated expression in the brain that have halted many clinical trials in the past. Our ability to increase endogenous levels of the PD-associated DJ-1 protein represents the first formal prove that synthetic SINEUPs may be a new class of nucleic acid-based drugs.

We are conscious that any potential application of SINEUPs in therapy will strictly depend on their efficient delivery. To this purpose we are currently exploring the preservation of their activity with different delivery systems and chemical modifications.

In summary, here we show first evidences that synthetic SINEUPs may represent a scalable platform for manipulating gene expression of single mRNA species within their physiological range in an array of cell lines.

SINEUPs may thus become a new tool for laboratory experiments, for protein manufacturing and for potential therapeutic intervention in vivo.

# Acknowledgments

We are indebted to all the members of the SG lab and to the SINEUP network (SISSA, University of Eastern Piedmont, RIKEN) for thought-provoking discussions. We thank Cristina Leonesi for technical support. We are grateful to SISSA technical and administrative staff, especially to J. Franzot, H. Krmac, Monica Sirk and Annalisa Sulli. This work was supported by the Italian Ministry of Education, University and Research (FIRB grant prot. RBAP11FRE9) to SG and FP.

# Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fncel. 2015.00174/abstract

Supplementary Figure 1 | Treatment with stressful stimuli does not increase SINEUP activity in transfected cells. HEK 293T/17 cells were transfected with pEGFP in combination with SINEUP-GFP (+SINEUP) or control plasmid (-SINEUP). After transfection, cells were treated with rapamycin or doxorubicin as indicated. Lysates were probed anti-GFP antibody. β-actin was used as loading control. Fold-induction was calculated on Western blot images normalized to β-actin and relative to empty control samples.

Supplementary Figure 2 | SINEUP RNA is detected in the cytoplasm of transfected cells. HEK 293T/17 cells were transfected with pEGFP in combination with SINEUP-GFP (+SINEUP). RNA was purified from separated

nuclear and cytoplasmic fractions. RNA was reverse transcribed and probed for SINEUP RNA and GFP mRNA, as indicated. Purity of nuclear and cytoplasmic fractions was monitored by qRT-PCR on precursor rRNA. Data were normalized to the level of GAPDH in each fraction and analyzed with the 11 Ct method. RNA levels in the cytoplasm were set to 1. Data indicate mean ± standard deviation and are calculated on 3 independent replicas.

Supplementary Figure 3 | SINEUP-increased targets can be detected with target-specific antibodies. HEK 293T/17 cells were transfected

# References

with pcDNA3-2XFLAG-TRAF6 in combination with SINEUP-FLAG (+SINEUP) or control plasmid (-SINEUP). Lysates were probed anti-TRAF6 antibody.

#### Supplementary Figure 4 | SINEUP-FLAG does not increase FLAG-TTRAP protein levels. HEK 293T/17 cells were transfected with

pcDNA3-2XFLAG-TTRAP in combination with SINEUP-FLAG (+SINEUP) or control plasmid (-SINEUP). Lysates were probed anti-FLAG antibody. SINEUP RNA and TTRAP mRNA were quantified by qRT-PCR with specific primers. Data indicate average ± stdev and are representative of *n* = 3 independent experiments.

**Conflict of Interest Statement:** Stefano Gustincich, Piero Carninci, Claudio Santoro, Michael H. Jones and Silvia Zucchelli declare competing financial interests as co-founders and members of TransSINE Technologies (www.transsine. com). Stefano Gustincich, Piero Carninci and Silvia Zucchelli are named inventors in patent issued in the US Patent and Trademark Office on SINEUPs and licensed to TransSINE Technologies. Michael H. Jones is CEO of Cell Guidance Systems, a company distributing SINEUPs as laboratory reagents. Stefano Gustincich and Piero Carninci are co-founders of PARKscreen, an Italian SME aimed to use and develop therapeutic SINEUPs.

Copyright © 2015 Zucchelli, Fasolo, Russo, Cimatti, Patrucco, Takahashi, Jones, Santoro, Sblattero, Cotella, Persichetti, Carninci and Gustincich. 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.

# Involvement of ELAV RNA-binding proteins in the post-transcriptional regulation of HO-1

#### **Marialaura Amadio1† , Giovanni Scapagnini 2,3\* † , Sergio Davinelli 2 , Vittorio Calabrese3,4 , Stefano Govoni 1 and Alessia Pascale1**

1 Department of Drug Sciences, Section of Pharmacology, University of Pavia, Pavia, Italy

2 Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy

3 Inter-University Consortium "SannioTech", Benevento, Italy

4 Department of Biomedical Sciences, University of Catania, Catania, Italy

#### **Edited by:**

Rosanna Parlato, Ulm University, Germany

#### **Reviewed by:**

Zsolt Radak, Semmelwies University, Hungary Pasquale Vito, Università del Sannio, Italy

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

Giovanni Scapagnini, Department of Medicine and Health Sciences, University of Molise, Via De Sanctis 86100, Campobasso, Italy e-mail<EMAIL_ADDRESS>†These authors have contributed equally to this work.

Heme oxygenase-1 (HO-1) is an inducible rate-controlling enzyme of heme catabolism. The cytoprotective function of HO-1 activity has been verified in multiple studies, and together with its by-products is considered a key component of the cellular stress response. The transcriptional induction of HO-1 has been largely studied in response to multiple forms of stressful stimuli but our understanding of HO-1 post-transcriptional control mechanisms in neuronal cells is currently lacking. In the present report we show the involvement of the RNA-binding proteins (RBPs) embryonic lethal abnormal vision (ELAV) in the regulation of HO-1 gene expression. Our study demonstrates a specific binding between HO-1 messenger RNA (mRNA) and ELAV proteins, accompanied by an increased expression of HO-1 at protein level, in a human neuroblastoma cell line treated with hemin. Clarifying the induction of HO-1 expression at post-transcriptional level may open therapeutic perspectives for treatments associated with the modulation of HO-1 expression.

**Keywords: heme oxygenase-1, ELAV, hemin, post-transcriptional regulation**

# **INTRODUCTION**

Heme oxygenase-1 (HO-1) is an intracellular enzyme that catalyzes the initial and rate-limiting step in the oxidative degradation of heme, and generates biliverdin, free iron (Fe2+), and carbon monoxide (CO; Calabrese et al., 2006). HO-1 is a heat shock protein (HSP32) and it is recognized as one of the major stress-inducible protein in mammalian cells (Maines, 1997). HO-1 activity can increase several-fold in response to a wide variety of stimuli that cause changes in the cellular redox state (Ryter and Choi, 2002). The products of heme metabolism such as bilirubin, ferritin and CO, mediate many of the anti-inflammatory and antioxidant effects associated with the potent cytoprotection provided by HO-1 enzymatic system (Kirkby and Adin, 2006). HO-1 expression is induced ubiquitously in response to oxidative challenges but brain tissue is particularly susceptible to free radical damage (Poon et al., 2004). Indeed, there is an increasing support that HO-1 plays a crucial protective role in the central nervous system (CNS), primarily in astrocytes and microglia/macrophages, particularly during aging and in several disease states where oxidative stress is implicated, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD; Calabrese et al., 2004). Although numerous published reports have demonstrated multiple beneficial effects of HO-1, its mechanism of action has not been completely elucidated. Expression of HO-1 is regulated essentially at transcriptional level, even though a posttranscriptional modulation of HO-1 messenger RNA (mRNA) in distinct cellular contexts has been described (Gozzelino et al., 2010). For instance, in human dermal fibroblasts hypoxia regulates HO-1 gene expression by a specific post-transcriptional mechanism: stabilization of mRNA (Kitamuro et al., 2003). Therefore, the regulation of HO-1 mRNA levels in response to cellular stress may be induced by both transcriptional and post-transcriptional events that act independently, and vary in function of the stress inducer (Leautaud and Demple, 2007). Although post-transcriptional control mechanisms are not yet fully understood, it is clear that they are key determinants in the regulation of mRNA stability and translation (Bolognani and Perrone-Bizzozero, 2008; Pascale et al., 2008; Keene, 2010). In particular, RNA-binding proteins (RBPs) regulate gene expression at post-transcriptional level and influence pre-mRNA processing as well as transport, localization, stability and translation of target mRNAs (Dreyfuss et al., 2002). RBPs are crucial in many aspects of cellular physiology and may play a direct role in the pathophysiology of neurodegenerative diseases (Pascale and Govoni, 2012; Perrone-Bizzozero and Bird, 2013; Romano and Buratti, 2013). Recent studies have identified hundreds of RBPs previously unknown (Castello et al., 2012) and a new and fascinating idea is that neurons have their own systems for regulating RNA metabolism, processing, localization, and expression (Darnell, 2013). In this context, embryonic lethal abnormal vision (ELAV) proteins are RBPs mostly expressed in neurons and post-transcriptional regulation in neuronal cells strongly depends on the control exerted by these RBPs (Pascale et al., 2008). In vertebrates, the ELAV (or Hu) family comprises the neuronspecific members HuB, HuC and HuD, and the ubiquitously expressed HuR (Colombrita et al., 2013). Moreover, emerging insights into the regulation by which cells establish patterns of gene expression suggest that post-transcriptional events influence inducible genes and signaling cascades such as HSPs and oxidative stress-activated pathways (Abdelmohsen et al., 2008; Amadio et al., 2008). HO-1 gene product and its mRNA abundance is controlled at various stages and it was reported that HO-1 induction by nitric oxide (NO) is regulated by the HuR/ELAV. Interestingly, the HuR/ELAV was found to associate with HO-1 mRNA, and this interaction increased following NO treatment (Kuwano et al., 2009). The HO-1 chemical inducer hemin is a highly reactive compound exhibiting pro-oxidant properties in several biochemical reactions. In the present study, we investigated, in SH-SY5Y human neuroblastoma cells, the expression of HO-1 mRNA and protein following hemin exposure and whether in these conditions HO-1 mRNA may represent a target of ELAV RBPs.

# **MATERIALS AND METHODS**

# **CELL CULTURES AND TREATMENTS**

The SH-SY5Y human neuroblastoma cells were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, L-glutamine (2 mM), nonessential amino acids (1 mM), and sodium pyruvate (1 mM) at 37◦C in an atmosphere of 5% CO2 and 95% humidity, as previously described (Racchi et al., 2003). The cells were exposed to the solvent (PBS) or to hemin (50 or 100 µM as reported in figure legends) for 2 h.

### **REVERSE-TRANSCRIPTION AND REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR)**

Total RNA was extracted from cells using the RNeasy Micro Kit (Qiagen, Milan, Italy). The reverse transcription was performed following standard procedures. PCR amplifications were performed using the LightCycler instrument (Roche Molecular Biochemicals) in the presence of QuantiTect SYBR Green PCR mix (Qiagen, Milan, Italy), with primers designed by using the PRIMER3 software.1 Primer sequences were as follows: HO-1, 5 0 -AGC AAC AAA GTG CAA GAT TCT GC-30 (forward); 50 - CAG CAT GCC TGC ATT CAC ATG-30 (reverse); product size: 161 bp; RPL6 (Ribosomal Protein L6), 50 - AGA TTA CGG AGC AGC GCA AGA TTG-30 (forward), 50 -GCA AAC ACA GAT CGC AGG TAG CCC-30 (reverse). RPL6 mRNA was chosen as the reference mRNA on which HO-1 was normalized because this RNA remained substantially stable during the treatments. Since RPL6 mRNA does not bear ARE sequences, it was also used as negative control in the immunoprecipitation experiments coupled with real-time quantitative polymerase chain reaction (qPCR).

### **IMMUNOPRECIPITATION**

Following treatment, SH-SY5Y cells were harvested and homogenized in a buffer [containing 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 50 mM 2-mercaptoethanol, 0.32 mM sucrose, and a protease inhibitor cocktail] by using a teflon/glass homogenizer. Immunoprecipitation was performed on total homogenates according to a previously published protocol (Amadio et al., 2009). Briefly, immunoprecipitation was carried out at room temperature for 2 h using 1 µg of anti-ELAV antibody (Santa Cruz Biotech, CA, USA) per 50 µg of proteins diluted with an equal volume of 2× Immunoprecipitation Buffer [2% Triton X-100, 30 mM NaCl, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 0.4 mM sodium vanadate, protease inhibitor cocktail and a RNAase inhibitor] in presence of 50 µl of protein A/G plus agarose beads (Santa Cruz Biotech, CA, USA) previously blocked with 5% BSA in PBS. The samples were finally subjected to RNA extraction and reverse transcription. The negative control was obtained in the same conditions, but in presence of an irrelevant antibody with the same isotype of the specific immunoprecipitating antibody. For binding assay, 100 µl of the immunoprecipitation mixes were collected from each sample and used as "input signal" to normalize the realtime qPCR data. For both HO-1 and RPL6, the mRNA content present in the immunoprecipitated pellet has been normalized on the mRNA amount present in the relative "input signal".

## **WESTERN BLOTTING**

Total lysate were diluted in 2× sodium dodecyl sulfate (SDS) protein gel loading solution, boiled for 5 min, separated by 12% SDSpolyacrylamide gel electrophoresis, and then processed following standard procedures. The antibodies anti-ELAV, anti-HO-1, and anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in TBST buffer [10 mM Tris-HCl, 100 mM NaCl, 0.1% (v/v) Tween 20, pH 7.5] containing 5% milk. The nitrocellulose membranes were processed with Pierce ECL Plus from Thermo Scientific (Rockford, IL, USA). The experiments were performed at least on three different cell preparations using α-tubulin to normalize data. Densitometric analysis was performed using the NIH Image software.2

# **DATA ANALYSIS**

For statistical analysis the GraphPad Instat statistical package (version 3.05 GraphPad software, San Diego, CA, USA) was used. The data were analyzed by analysis of variance (ANOVA) followed, when significant, by an appropriate *post hoc* comparison test, as indicated in figure legends. Differences were considered statistically significant when *p* values ≤ 0.05.

# **RESULTS**

# **HEMIN INCREASES HO-1 mRNA LEVELS IN SH-SY5Y CELLS**

To determine whether hemin treatment affects HO-1 expression in human neuroblastoma SH-SY5Y cells, we first measured HO-1 mRNA levels in total homogenates of cells treated with hemin (100 µM) for 2 h + 4 h recovery. Real-time qPCR data demonstrated a marked increase of HO-1 mRNA level in 2 h hemintreated samples followed by 4 h recovery as compared to the other samples (**Figure 1**). RPL6 mRNA level remained substantially stable during the experiments (data not shown). These data reveal that in SH-SY5Y cells hemin treatment up-regulates the expression of HO-1 mRNA.

1http://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm

2http://rsb.info.nih.gov/nih-image

#### **ASSOCIATION OF HO-1 mRNA WITH ELAV RBPs**

To test the hypothesis that ELAV RBPs interact with HO-1 mRNA following hemin treatment, we performed an immunoprecipitation assay on SH-SY5Y cells, which express all the four isoforms of ELAV proteins, followed by real-time qPCR. As shown in **Figure 2**, the association of ELAV RBPs with HO-1 mRNA occurs; indeed, we found that the amount of HO-1 transcript bound by ELAV proteins in the total homogenate is increased following 2 h hemin stimulus + 4 h recovery (**Figure 2**). Moreover, the housekeeping RPL6 mRNA was almost undetectable in the same immunoprecipitated pellets (**Figure 2**), confirming the existence of a specific binding between ELAV proteins and HO-1 mRNA.

### **HEMIN TREATMENT UPREGULATES HO-1 PROTEIN LEVELS IN A CONCENTRATION-DEPENDENT MANNER**

We finally measured HO-1 protein levels by Western blotting, finding an increase after 2 h hemin exposure followed by 4 h recovery (**Figure 3**). Interestingly, in basal conditions, HO-1 protein content is almost undetectable and its increase is proportional to hemin concentration, reaching statistical significance only at 100 µM concentration (**Figure 3**). In the same samples we also measured ELAV proteins levels, finding they are not modified following hemin stimulus (not shown).

## **DISCUSSION**

HO-1 is one of the main players of the endogenous antioxidant responses and since it provides cytoprotection against various neurotoxic insults, it is crucial to elucidate how HO-1 gene expression is regulated. A comprehensive knowledge regarding HO-1 expression in human neural cells is still lacking. Although most studies have focused on the molecular mechanisms responsible for the cytoprotective effects of HO-1, the importance of post-transcriptional processes in the modulation of HO-1 gene expression has been greatly underestimated. Furthermore, there is an emerging consensus that neuronal transcripts are

differentially processed in the brain than in other tissues (Darnell, 2013). Interestingly, it was recently demonstrated that RBPs and non-coding RNAs are critical components underlying the post-transcriptional mechanisms for the coordinate regulation of mRNA expression in neuronal systems (Loya et al., 2010). Human ELAV RBPs are involved in the post-transcriptional control of several early-responsive genes such as MYC, FOS and cytokines (Keene, 2007; Papadopoulou et al., 2013). Since HO-1 system is a promising approach to treat specific disorders and its induction has significant consequences in the CNS, the present study explores the role of ELAV proteins in the regulation of HO-1 expression at both mRNA and protein levels. In particular, we treated human neuroblastoma SH-SY5Y cells with hemin, a pro-oxidant molecule. Although HO-1 is activated by various electrophilic compounds including polyphenols (Davinelli et al., 2013), hemin is one of the most effective stressors in terms of reactive oxygen species (ROS) production and it is widely used to study HO-1 function. Therefore, we chose hemin as a challenge for our experiments. However, it is essential to point out that at elevated concentrations hemin may contribute to cell injury by cytotoxic effects, although strengthening endogenous defense against oxidative stress, at nontoxic concentrations its therapeutic

blotting of HO-1 and α-tubulin in control (CTR) and hemin-treated SHSY5Y cells. Cells were exposed to either 50 or 100 µM hemin for 2 h followed by 4 h recovery (rec). **(Lower)** Mean gray levels ratios (mean ± S.E.M.) expressed as percentages with respect to control (100%) of HO-1/α-tubulin immunoreactivities measured by Western blotting. \*p < 0.05; \*\*p < 0.005, Tukey-Kramer Multiple comparisons test, n = 3.

potential has been reported in multiple acute injury models, including those at brain level (Lu et al., 2014). In our study, we found that HO-1 expression is induced in 100 µM hemin-treated neuronal cells, as demonstrated by real time qPCR and Western blotting experiments (**Figures 1**, **3**). This finding is consistent with previous studies showing the activation of HO-1 by hemin in human neuroblastoma cells (Nakaso et al., 2003). Interestingly, and in accordance with previous evidence in rat hippocampal neurons (Scapagnini et al., 2004), we observed that, in human SH-SY5Y neuroblastoma cells, HO-1 protein expression is almost absent in basal conditions and it is strongly induced by hemin in a concentration-dependent manner (**Figure 3**). However, we cannot exclude that the sensitivity of Western blotting technique is not sufficient to detect very low amount of protein as in control cells. The hemin-induced increase of HO-1 protein may be due to a positive regulation of HO-1 at post-transcriptional level. Indeed, our results shows a specific association between ELAV and HO-1 mRNA (**Figure 2**), suggesting that this binding may have potential consequences for HO-1 protein expression. Our hypothesis is that HO-1 is present as mRNA in control cells so that, as many early response genes, it can be rapidly translated following a specific stress stimulus, such as hemin exposure, thus contributing to the physiological cellular response.

Although it was well-established that HO-1 is highly inducible by a large number of stressful stimuli such as heme or certain other metalloporphyrins, the regulation of gene expression and

induction of HO-1 is complicated by the fact that its regulatory response is not restricted to heme or other physical and chemical factors. A purpose of this study was to bridge the significant gap in understanding the regulation of HO-1 induction in neural cells. Previous evidence from our group showed that the ELAV RBPs are involved in regulating the post-transcriptional fate of HSP70 and SOD-1 mRNA following H2O2-mediated oxidative stress in SH-SY5Y cells (Amadio et al., 2008; Milani et al., 2013). HSP70, SOD-1 and HO-1 modulate crucial defensive mechanisms for neurons exposed to an oxidant challenge. Consistently with the data reported for the other genes, the results described in this report suggest a novel role for ELAV RBPs in regulating, post-transcriptionally, HO-1 expression in SH-SY5Y neuronal cells, as schematically represented in **Figure 4**. Future studies are needed to identify whether this interaction is a characteristic feature of HO-1 induction during the cellular stress response.

# **REFERENCES**

antioxidative proteins by hemin in human neuroblastoma cells. *FEBS Lett.* 546, 181–184. doi: 10.1016/s0014-5793(03)00517-9

**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: 20 July 2014; accepted: 17 December 2014; published online: 15 January 2015*.

*Citation: Amadio M, Scapagnini G, Davinelli S, Calabrese V, Govoni S and Pascale A (2015) Involvement of ELAV RNA-binding proteins in the post-transcriptional regulation of HO-1. Front. Cell. Neurosci. 8:459. doi: 10.3389/fncel.2014.00459 This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2015 Amadio, Scapagnini, Davinelli, Calabrese, Govoni and Pascale. 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*.

# RTP801/REDD1: a stress coping regulator that turns into a troublemaker in neurodegenerative disorders

# **Mercè Canal , Joan Romaní-Aumedes , Núria Martín-Flores , Víctor Pérez-Fernández and Cristina Malagelada \***

Department of Pathological Anatomy, Pharmacology and Microbiology, Faculty of Medicine, University of Barcelona, Barcelona, Catalonia, Spain

#### **Edited by:**

Rosanna Parlato, Ulm University, Germany

#### **Reviewed by:**

Hermona Soreq, The Hebrew University of Jerusalem, Israel Davide Pozzi, Humanitas Research Hospital, Italy

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

Cristina Malagelada, Department of Pathological Anatomy, Pharmacology and Microbiology, Faculty of Medicine, University of Barcelona, Casanova 143, Barcelona, Catalonia 08036, Spain e-mail<EMAIL_ADDRESS>
Mechanistic target of Rapamycin (mTOR) pathway regulates essential processes directed to preserve cellular homeostasis, such as cell growth, proliferation, survival, protein synthesis and autophagy. Importantly, mTOR pathway deregulation has been related to many diseases. Indeed, it has become a hallmark in neurodegenerative disorders, since a fine-tuned regulation of mTOR activities is crucial for neuron function and survival. RTP801/REDD1/Dig2 has become one of the most puzzling regulators of mTOR. Although the mechanism is not completely understood, RTP801 inactivates mTOR and Akt via the tuberous sclerosis complex (TSC1/TSC2) in many cellular contexts. Intriguingly, RTP801 protects dividing cells from hypoxia or H2O2-induced apoptosis, while it sensitizes differentiated cells to stress. Based on experimental models of Parkinson's disease (PD), it has been proposed that at early stages of the disease, stress-induced RTP801 upregulation contributes to mTOR repression, in an attempt to maintain cell function and viability. However, if RTP801 elevation is sustained, it leads to neuron cell death by a sequential inhibition of mTOR and Akt. Here, we will review RTP801 deregulation of mTOR in a context of PD and other neurodegenerative disorders.

**Keywords: RTP801, REDD1, mTOR, Akt, stress, neurodegeneration, neuron, Parkinson's disease**

#### **RTP801 OVERVIEW**

RTP801 (also known as REDD1 or Dig2) is a protein encoded by the stress responsive gene DNA-damage-inducible transcript 4 (*DDIT4*). It was initially identified and cloned in 2002 by two different groups simultaneously.

Shoshani et al. (2002) screened for hypoxia-regulated genes in rat C6 glioma cells and they identified a highly up-regulated gene responsive to HIF-1, involved in the regulation of cellular reactive oxygen species (ROS). It was designated *RTP801* (Shoshani et al., 2002).

Concurrently, Ellisen et al. (2002) cloned a gene induced after DNA damage and during embryogenesis in a p53 and p63 dependent manner. This gene was involved in the regulation of ROS and was alternatively named *REDD1*, for regulated in development and DNA damage responses one (Ellisen et al., 2002).

Later in 2003, Wang et al. discovered Dig2 (for dexamethasone-induced gene 2), the mouse homolog of *RTP801/REDD1* in an oligonucleotide microarray analysis from dexamethasone-treated murine lymphoma T cells (Wang et al., 2003).

Further studies displayed *DDIT4* as a rapidly upregulated gene under multiple cellular stresses, such as heat shock (Wang et al., 2003), ionizing radiation (Ellisen et al., 2002), hypoxia (Shoshani et al., 2002; Brugarolas et al., 2004) and energy depletion (Sofer et al., 2005). Moreover other chemical molecules also upregulated *DDIT4* expression, such as dopaminergic neurotoxins 6-hydroxydopamine, MPTP/MPP+ and rotenone (Malagelada et al., 2006), endoplasmatic reticulum (ER) stress inducers tunicamycin and thapsigargin (Wang et al., 2003; Whitney et al., 2009), DNA damage agent etoposide (Wang et al., 2003) and arsenite (Lin et al., 2005b).

RTP801, as a 232 aminoacids protein, is ubiquitously expressed at low levels in numerous human adult tissues (Shoshani et al., 2002). RTP801 localizes in the cytoplasm, the nucleus (Ellisen et al., 2002; Lin et al., 2005b; Michel et al., 2014) and in the membranes (DeYoung et al., 2008; Michel et al., 2014). Besides, a small fraction of RTP801 was detected in the mitochondria in HEK293T cells (Horak et al., 2010) and in *RGC*-*5* retinal ganglion cell line (del Olmo-Aguado et al., 2013).

There is a related human transcript called RTP801L (RTP801 like) or REDD2 that displays ∼50% sequence identity to RTP801 and has similar functions (Ellisen et al., 2002; Corradetti et al., 2005).

Besides humans, RTP801 is also present in other organisms such as rat, mouse and *Xenopus*. In *Drosophila* it has two related orthologs called Scylla and Charybdis (Reiling and Hafen, 2004).

No functional motifs or structural domains could be identified from RTP801 amino acid sequence analysis and, to date, the entire crystal structure has not been solved. Indeed, only one group crystallized a segment containing aminoacids 89–226 with a deletion of the hydrophobic region 200FLPGF204 of the human RTP801 protein (Vega-Rubin-de-Celis et al., 2010). Their work has given new insights into RTP801 structure. They found that RTP801 presents a unique topology characterized by a two-layered α/β sandwich with a psi-loop motif. Furthermore, a surface patch formed by highly conserved residues was found to be critical for its function. It is formed by two separated regions, 138EPCG141 and 218KKKLYSSE225, that are contiguous in the three-dimensional structure (Vega-Rubin-de-Celis et al., 2010). Importantly, the stretch of three lysines 218KKK220 is necessary for RTP801 to localize in both mitochondria (Horak et al., 2010) and plasma membrane (Michel et al., 2014).

The key function of RTP801 is its ability to inactivate mTOR (Brugarolas et al., 2004; Corradetti et al., 2005), a master regulator kinase that integrates extracellular signals with intracellular responses to nutrients, growth factors or stress. Indeed, mTOR regulation has a crucial role in development, cancer or in neural survival and plasticity (Hoeffer and Klann, 2010; Laplante and Sabatini, 2012).

RTP801 has a dual role depending on the cellular context, meaning that in proliferating non differentiated cells, RTP801 is anti-apoptotic, and in non-dividing differentiated cells like neurons, RTP801 is pro-apoptotic (Shoshani et al., 2002; Malagelada et al., 2006). This dual function could be nicely observed in a study of rat cortical neurogenesis, where RTP801 controlled neuroprogenitors proliferation and neuronal differentiation. In cortical neuroprogenitors RTP801 was elevated without being toxic. On the contrary, newborn and mature neurons showed lower levels of RTP801. Indeed, if RTP801 elevation was sustained in these differentiating neurons it became pro-apoptotic (Malagelada et al., 2011). How RTP801 can trigger these dual actions based on the cell context is not completely understood and requires further investigation.

In the last decade the role of mTOR in neural cells has become very relevant. Indeed, in the nervous system mTOR controls crucial processes such as protein translation, longlasting synaptic plasticity and survival via Akt (Tang et al., 2002; Cammalleri et al., 2003; Malagelada et al., 2008). Evidence suggests that mTOR deregulation is involved in neurodegeneration, and therefore the role of RTP801 has emerged as an object of study. In this sense, it will be crucial to understand the fine balance between RTP801 as a stress-coping protein and RTP801 as a pro-apoptotic effector in neurological disorders. Understanding these complex mechanisms will help to design successful therapeutic strategies to halt or, at least, delay neurodegeneration.

# **HOW IS RTP801 UPREGULATED?**

Previous studies suggest that RTP801 toxicity in neurons is proportional to its protein levels (Malagelada et al., 2010; Ota et al., 2014). RTP801 protein increase can be the end point of two different processes: (1) as a result of gene activation by cellular stress (Ryu et al., 2005; Malagelada et al., 2006); and (2) a defective RTP801 degradation (Romaní-Aumedes et al., 2014). Here we will describe the transcription factors responsible for *DDIT4* gene induction, the microRNAs (miRNAs) that regulate its translation and the post-translational events in charge of regulating RTP801 protein levels.

# **RTP801 TRANSCRIPTIONAL REGULATION**

The variety of transcription factors able to induce *DDIT4* gene expression in response to different stressors illustrates the complexity of its regulation. In fact, one feature of the regulation of RTP801*/DDIT4* is its rapidity, crucial to activate the coping mechanisms of the cell in response to the hostile environment. For example, hypoxia upregulates RTP801 expression via HIF-1, since the *DDIT4* gene contains a HRE (hypoxia-response element) in the promoter (Shoshani et al., 2002; Brugarolas et al., 2004). Another hypoxia-mimetic agent, cobalt chloride (CoCl2), needs co-activation of HIF-1 and Sp1 to induce RTP801 (Jin et al., 2007).

Deoxyribonucleic acid (DNA) damaging agents, including ionizing radiation and the DNA alkylating agent methyl methane sulfonate (MMS) also boosted RTP801 expression (Ellisen et al., 2002; Lin et al., 2005a). Ionizing radiation induced RTP801 in a p53-dependent manner in mouse embryonic fibroblasts (MEFs; Ellisen et al., 2002). DNA-damage-inducible transcript 4 transcription was also enhanced by MMS in human keratinocytes via Elk-1 and CCAAT/enhancer-binding protein (C/EBP) in a p53-independent manner (Lin et al., 2005a). Furthermore, RTP801 has also been identified as a transcription target of Elk-1 and C/EBP in response to arsenic-induced ROS (Lin et al., 2005b).

Endoplasmatic reticulum (ER) stress caused by tunicamycin or thapsigargin upregulated RTP801 via activating transcriptional factor 4 (ATF4; Jin et al., 2009; Whitney et al., 2009). ATF4 was also identified as a transcription factor for RTP801 in response to oxidative stress induced by hydrogen peroxide (Jin et al., 2009). Interestingly, ATF4 has a protective role in cellular models of Parkinson's disease (PD) by modulating the levels of the E3 ligase parkin (Sun et al., 2013).

Other transcription factors have been described for *DDIT4* gene regulation like the nuclear factor of activated T-cell c3 (NFAT c3; Zhou et al., 2012) or PLZF in spermatogonial progenitors (Hobbs et al., 2010).

It is noteworthy that all these stressors, via different transcription factors, elevate RTP801 with a common objective to inactivate mTOR. This common response to stressors corroborates the complexity of the integration of the stress signals to modulate mTOR effectively.

Many other stress responsive genes with pro-apoptotic functions are also upregulated in parallel with DDIT4. However, due to space limitations, they will not be reviewed in this text (reviewed in Fulda et al., 2010).

### **RTP801 TRANSLATIONAL REGULATION**

MicroRNA are negative regulators of gene expression and can function as tumor suppressors or oncogenes. To date, at least three miRNAs have been described as regulators of RTP801/REDD1 expression in a context of tumorigenesis.  Another miRNA, the miR-221, stimulates hepatocarcinogenesis by downregulating RTP801 expression (Pineau et al., 2010). Furthermore, Micro-RNA30c down-regulates REDD1 expression in human hematopoietic and osteoblast cells after gamma-irradiation (Li et al., 2012).

To our knowledge, no miRNA that modulates RTP801 expression in a context of neurodegeneration has been described. In a near future, miRNAs along with the long non-coding RNAs may have a relevant impact in the regulation of RTP801 levels and function.

# **RTP801 POST-TRANSLATIONAL REGULATION**

Post-translational modifications like phosphorylation, acetylation, ubiquitination or myristoylation, have an important impact in protein stability, function and cellular localization. Indeed, cellular stresses can also affect turnover of many proteins, including RTP801.

Apart from its rapid gene induction under stress, RTP801 proteostasis will also determine its stability, and therefore, its regulatory function towards mTOR.

Related to that, RTP801 mRNA (Wang et al., 2003) and RTP801 protein (Kimball et al., 2008; Katiyar et al., 2009; Malagelada et al., 2010) half-lives are significantly short, revealing that RTP801 is an extremely unstable protein with a fine-tuned post-translational regulation.

One of the modifications that will lead to a rapid protein turnover is ubiquitination. In fact, RTP801 is poly-ubiquitinated and targeted for the ubiquitin-proteasome system (UPS; Katiyar et al., 2009; Romaní-Aumedes et al., 2014).

To date, only three E3 ring ligases have been identified to poly-ubiquitinate RTP801. The first one described was CUL4A-DDB1-ROC1-β-TRCP E3 ligase complex. The complex ubiquitinated RTP801 and targeted it for proteasomal degradation, in a GSK3β-phosphorylation-dependent manner (Katiyar et al., 2009).

The second E3 ubiquitin ligase for RTP801 was HUWE1/MULE that modulated RTP801 protein levels although this regulation seemed to be UPS-independent (Tan and Hagen, 2013).

So far, the role of both ligases in regulating RTP801 in neurodegenerative disorders has not been elucidated.

In our recent work, we found that parkin RING E3 ligase poly-ubiquitinates RTP801 to mediate its UPS degradation. Based on the results obtained in cellular and animal models and in samples from human parkin mutant carriers, we proposed that RTP801 elevation due to parkin loss-of-function in both parkin mutants and in idiopathic PD might contribute importantly to neurodegeneration (Romaní-Aumedes et al., 2014).

In a near future, other ligases will eventually be proved to ubiquitinate RTP801, due to its central role in regulating mTOR.

# **RTP801 INACTIVATES mTOR VIA TUBEROUS SCLEROSIS COMPLEX**

Under stress conditions downregulation of mTOR activity caused by hypoxia, energy stress or exposure to dopaminergic neurotoxins required the expression of RTP801 and an intact TSC1/TSC2 tumor suppressor complex (Brugarolas et al., 2004; Sofer et al., 2005; Malagelada et al., 2006). Interestingly, despite the clear necessity of the intact TSC1/2 complex for RTP801 to downregulate mTOR, RTP801 does not seem to interact physically with either TSC1 or TSC2 (Vega-Rubin-de-Celis et al., 2010).

Tuberous sclerosis complex (TSC) is a heterodimer formed by two proteins, the tuberous sclerosis tumor suppressors TSC1 and TSC2. TSC2 has a catalytic function as a GTPase activating protein (GAP), and it acts toward the small GTPase Rheb, an upstream positive mTOR regulator (Inoki et al., 2003; Tee et al., 2003). TSC2 can be phosphorylated by several kinases in response to upstream signals, and they will modify its regulation towards mTORC1 (reviewed in Ma and Blenis, 2009). Akt can phosphorylate TSC2 in response to growth factors (Inoki et al., 2002). This event is thought to favor TSC2 binding to protein 14-3-3, instead to TSC1, leading to TSC2 inhibition, to finally activate mTORC1 (Cai et al., 2006).

Regarding the modulatory role of 14-3-3 towards TSC2, DeYoung et al. (2008) proposed a molecular mechanism by which RTP801 regulates TSC1/2-mTOR signaling involving 14- 3-3. Other studies also reported interaction between RTP801 and 14-3-3 proteins by co-immunoprecipitation experiments (Favier et al., 2010; Hernández et al., 2011; Pieri et al., 2014).

However, this RTP801 direct binding to 14-3-3 has been questioned by others (Vega-Rubin-de-Celis et al., 2010). The supposed 14-3-3 binding motif in RTP801 (133RLAYSEP139) is not conserved within species and the crystallized RTP801 structure analysis does not reveal any established mode for 14-3-3 binding. Thus, the inhibitory mechanisms of RTP801 towards mTOR need to be further investigated.

# **RTP801 UPREGULATION IN NEURODEGENERATION**

The etiology of PD and many other neurodegenerative disorders involves both environmental factors and genetic predisposition. Indeed, exposure to several environmental toxins such as pesticides and metals has been implicated in its pathogenesis (Migliore and Coppede, 2009; Cannon and Greenamyre, 2011; Baltazar et al., 2014).

Arsenic is a heavy metal that has been linked to neurotoxicity and carcinogenesis in humans by a mechanism involving ROS production (reviewed in Qian et al., 2003). Interestingly, arsenite induced RTP801 transcription (Lin et al., 2005b).

Moreover, extensively used dithiocarbamate pesticides Maneb (MB) and Mancozeb (MZ) induced DNA damage and elevated RTP801 mRNA and protein expression (Cheng et al., 2014). The toxicity mechanism of these pesticides has been linked to NFκB activation, revealing a cross-talk between RTP801 and NF-κB (Cheng et al., 2014).

In line with this, RTP801 was also identified as an amyloidβ-peptide (Aβ) responsive gene. Amyloid-beta is a neurotoxic molecule and the main component of senile plaques in Alzheimer's disease (AD; Kim et al., 2003).

Dopaminergic neurotoxins also upregulated RTP801 in cellular and animal models. Specifically, RTP801 was upregulated at both transcriptional and protein level in neuronal PC12 cells treated with PD mimetic toxins 6-OHDA, MPP+ and rotenone, and it was also induced in neurons of MPTP-treated mice and in Substantia Nigra pars compacta (SNpc) degenerating neurons of PD patients. Furthermore, RTP801 knockdown with short hairpin RNAs (shRNAs) protected the cultures from PD mimetic toxins (Malagelada et al., 2006).

Ubiquitin-proteasome system deregulation has been implicated in neurodegenerative disorders (Keller et al., 2000; Jana et al., 2001; McNaught et al., 2003). Accumulations of misfolded or aggregated proteins may hamper cellular functions and eventually lead to neuronal death (reviewed in Tanaka and Matsuda, 2014). RTP801 brief protein half-life suggests that UPS malfunction would affect RTP801 protein levels. Therefore it is logical to suggest that a proper RTP801 degradation might be crucial for neuron function and survival.

Thus, chronic and progressive RTP801 elevation could become a hallmark of neurodegenerative disorders due to a combination of stress-induced gene activation and UPS malfunction.

# **THE ROLE OF RTP801 IN NEURODEGENERATIVE DISORDERS**

RTP801 behaves like many other stress-induced genes, where a small increase is beneficial but a chronic and sustained increase is detrimental for the neuron. Interestingly, since RTP801 is able to inactivate protein translation, via mTOR, and survival, via Akt (Malagelada et al., 2006, 2008, 2010), its pro-apoptotic role may be relevant to other neurodegenerative diseases.

In PD, RTP801 elevation was initially identified in cellular and animal models of the disease. These results were further confirmed in nigral neurons from both idiopathic PD and mutant parkin human brains, meaning that RTP801 may have a relevant role in the disease itself (Malagelada et al., 2006).

Since RTP801 elevation is necessary and sufficient to trigger neuronal death, it is logical to think that the upregulation in the SN from PD human brains might be detrimental for the nigral neurons. Interestingly RTP801 induces cell death through a mechanism involving TSC1/2 and mTOR repression (Malagelada et al., 2006). Indeed, RTP801 repression of mTOR led also to the suppression of the phosphorylation of the neuronal survival kinase Akt (Malagelada et al., 2008), reviewed in (Greene et al., 2011). This negative regulation of Akt was also observed in nigral neurons from PD human brains.

Based on these results, we proposed a mechanism to explain RTP801 contribution to neurodegeneration in PD, where low levels of RTP801 would help neurons to cope with stress, but if this inactivation were sustained in time, it would impair neuronal function and survival. At the latest stage, and when the critical threshold of mTOR/Akt inactivation is surpassed, neuronal death will occur (Malagelada et al., 2006, 2008; see **Figure 1**). This proposed mechanism is supported by the observation of elevated RTP801 and diminished Akt phosphorylation in nigral neurons of PD brains (Malagelada et al., 2006; Romaní-Aumedes et al., 2014).

To extend this working model, one possibility would be that at early stages of many neurodegenerative disorders, RTP801 induction by cellular stresses would contribute to mTOR depression in an attempt to preserve neuron function and viability. However, at more advanced disease stages, when RTP801 upregulation is prolonged in time, it can eventually promote neurodegeneration and neuronal death through mTOR and pro-survival kinase Akt inactivation, as it has been proposed in the PD studies.

In AD an elevation of both RTP801/REDD1 gene and protein was observed in lymphocytes from AD patients compared to age-matched controls. Damjanac et al. (2009) showed that Double-stranded RNA-dependent protein kinase (PKR), a cognitive decline biomarker with an impairing protein translation function, by phosphorylating p53, activated REDD1 gene transcription. It could be interesting to explore whether this mechanism can be observed also in hippocampal neurons or in AD murine models.

Recently, RT801 has been determined crucial for stressmediated synaptic loss and depressive behavior. Chronic unpredictable stress increased RTP801 mRNA and protein levels in rat prefrontal cortex (PFC). This RTP801 upregulation was coincident with a reduced phosphorylation of mTORC1/2 signaling targets S6K, 4EBP1, and Akt. In agreement, RTP801 was elevated in postmortem PFC of patients with major depressive disorder compared to psychiatrically healthy controls. RTP801 knockout mice were resilient to the synaptic and behavior deficits caused by stress, while RTP801 overexpression in rat PFC was sufficient to promote neuronal atrophy and depressive behavior (Ota et al., 2014).

Further investigations are needed to elucidate the role of RTP801 in many other neurological disorders. It will be interesting to explore whether RTP801 levels are upregulated and whether they correlate with the degree of mTOR/Akt repression. Also, it will be important to determine whether RTP801 is promoting neuron death through the same mechanism proposed as in PD models.

# **RTP801 AS A POTENTIAL THERAPEUTIC TARGET IN NEUROLOGICAL DISORDERS**

Neurodegenerative disorders are characterized by neuronal death of specific subpopulations, such as loss of SN dopaminergic neurons in PD (reviewed in Dauer and Przedborski, 2003). However, current PD therapies ameliorate symptoms but do not prevent neuronal death. Thus, it is essential to investigate the mechanisms that underlie neuronal death and identify targets involved in the pathophysiology (reviewed in Levy et al., 2009). Based on several studies, RTP801 could become one such potential therapeutic target (Brafman et al., 2004; Malagelada et al., 2010; Tarazi et al., 2014).

RTP801 promotes cell death by sequentially inactivating mTOR and Akt (Malagelada et al., 2006, 2008). Thus, compounds that can restrain RTP801 expression or modulate the mTOR/Akt pathway may become therapeutic and delay neurodegeneration and neuronal cell death in PD (reviewed in Tarazi et al., 2014).

One such compound is 8-methyl-6-phenoxy-2-(tetrahydropyran-4-ylamino)pyrido[2,3-d]pyrimidin-7-one (FLZ), a synthetic squamoside derivative from a Chinese herb that protected dopaminergic neurons from apoptosis triggered by MPP+ and 6-OHDA (Zhang et al., 2007a,b). Interestingly, FLZ neuroprotective actions in PD involved activation of Akt/mTOR signaling pathway and inhibition of RTP801 expression (Bao et al., 2012, 2014).

Another promising compound is rapamycin, an allosteric inhibitor of some but not all mTOR activities. Rapamycin conferred neuroprotection in both cellular and animal models of PD (Tain et al., 2009; Dehay et al., 2010; Malagelada et al., 2010). The results obtained support the hypothesis in which rapamycin

blocks RTP801 translation and, as a consequence, it mitigates mTOR repression leading to Akt phosphorylation maintenance at a site critical for its pro-survival activity (Malagelada et al., 2010). In contrast, Torin 1, an inhibitor of all mTOR actions since it blocks the ATP-binding site, was not protective and promoted Akt dephosphorylation and neuron death. So, rapamycin protection derives from its partial suppression of certain mTOR actions due to its allosteric properties (Malagelada et al., 2010).

Nonetheless, further investigations are required to evaluate the potential therapeutic role of these two agents in PD.

In animal and cellular models of cerebral ischemia, Ligustilide, a major active agent of Radix Angelicae Sinensis, the root of a chinese herb called Danngui, is neuroprotective by inhibiting RTP801 expression, in addition to promote Erythropoietin transcription via extracellular-signal-regulated kinases (ERK) signaling pathway (Wu et al., 2011).

The most advanced example of RTP801 as a therapeutic target is found in retinopathies. RTP801 has an important role in the pathogenesis of retinopathies, since the absence of RTP801 expression in a mouse model attenuated the development of the disease (Brafman et al., 2004). PF-04523655, a 19-ribonucleotide siRNA designated to inhibit RTP801 transcription is currently in clinical trials for retinopathy treatment (Lee et al., 2012; Nguyen et al., 2012a,b; Rittenhouse et al., 2014).

In summary, RTP801 is one clear example of a protein that is upregulated to cope with cellular stress although its sustained progressive elevation leads to neuron degeneration and death. RTP801 progressive elevation and its inhibitory function towards pro-survival kinases mTOR and Akt could explain its role in several neurodegenerative diseases.

# **ACKNOWLEDGMENTS**

Supported in part by grants from the Spanish Ministry of Science and Innovation (SAF2010-21058), the PEOPLE Programme— Marie Curie Actions, European Community (PIRG08-GA-2010- 276957). Supported also by the crowdfunding campaign "SOS recerca en Parkinson" via Goteo.org, Portal d'Avall, S. L. and the app "Mememtum: early detection of neurological disorders". Illustrations were designed by Laura Olivares-Boldú.

# **REFERENCES**

Laplante, M., and Sabatini, D. M. (2012). mTOR signaling in growth control and disease. *Cell* 149, 274–293. doi: 10.1016/j.cell.2012.03.017

**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: 26 July 2014; accepted: 17 September 2014; published online: 02 October 2014*.

*Citation: Canal M, Romaní-Aumedes J, Martín-Flores N, Pérez-Fernández V and Malagelada C (2014) RTP801/REDD1: a stress coping regulator that turns into a troublemaker in neurodegenerative disorders. Front. Cell. Neurosci. 8:313. doi: 10.3389/fncel.2014.00313*

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

*Copyright © 2014 Canal, Romaní-Aumedes, Martín-Flores, Pérez-Fernández and Malagelada. 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*.

# Legal but lethal: functional protein aggregation at the verge of toxicity

# **Angelika Falsone and S. Fabio Falsone \***

Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria

#### **Edited by:**

Rosanna Parlato, Ulm University, Germany

#### **Reviewed by:**

Daniel Kaganovich, Hebrew University of Jerusalem, Israel, Israel Robert Weissert, University of Regensburg, Germany

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

S. Fabio Falsone, Institute of Pharmaceutical Sciences, University of Graz, Schubertstr. 1, 8010 Graz, Austria e-mail<EMAIL_ADDRESS>
Many neurodegenerative disorders are linked to irreversible protein aggregation, a process that usually comes along with toxicity and serious cellular damage. However, it is emerging that protein aggregation can also serve for physiological purposes, as impressively shown for prions. While the aggregation of this protein family was initially considered exclusively toxic in mammalians organisms, it is now almost clear that many other proteins adopt prion-like attributes to rationally polymerize into higher order complexes with organized physiologic roles. This implies that cells can tolerate at least in some measure the accumulation of inherently dangerous protein aggregates for functional profit. This review summarizes currently known strategies that living organisms adopt to preserve beneficial aggregation, and to prevent the catastrophic accumulation of toxic aggregates that frequently accompany neurodegeneration.

**Keywords: amyloids, prions, proteotoxicity, neurodegenerative diseases, proteostasis regulators**

# **INTRODUCTION**

Low structural complexity is at the basis of highly diversified molecular recognition, whereby one flexible protein region can bind to various heterogeneous ligands by conformational adaptation. Proteins can functionally benefit from binding promiscuity for key regulatory processes such as signal transduction, transcription, RNA processing and translation. Proteins situated on intersecting hubs of different pathways can undergo multifunctional interactions, functioning as molecular switches by means of conformational variability. However, the benefits of conformational freedom come along with the menace of protein misfolding and multifunctional failure. Although each living organism invests conspicuous amounts of energy for the rescue or elimination of misfolded polypeptides, a possible inability of the cell to cope with misfolded polypeptides inevitably leads to a massive functional destabilization, whereby proteins can either lose their original function (loss-of-function), or they acquire an improper, and therefore mostly lethal function (gain-of-function), eventually aggregating after the complete collapse of folding and clearance pathways.