s2fieldsofstudy: Biology Psychology Medicine

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Sex influences susceptibility to methamphetamine cardiomyopathy in mice

Abstract In this study, we created a mouse model of methamphetamine cardiomyopathy that reproduces the chronic, progressive dosing commonly encountered in addicted subjects. We gradually increased the quantity of methamphetamine given to C57Bl/6 mice from 5 to 40 mg/kg over 2 or 5 months during two study periods. At the fifth month, heart weight was increased, echocardiograms showed a dilated cardiomyopathy and survival was lower in males, with less effect in females. Interestingly, these findings correspond to previous observations in human patients, suggesting greater male susceptibility to the effects of methamphetamine on the heart. Transcriptional analysis showed changes in genes dysregulated in previous methamphetamine neurological studies as well as many that likely play a role in cardiac response to this toxic stress. We expect that a deeper understanding of the molecular biology of methamphetamine exposure in the heart will provide insights into the mechanism of cardiomyopathy in addicts and potential routes to more effective treatment.

Introduction
Methamphetamine (Meth) is a highly toxic and addictive drug that stimulates the central nervous system by triggering the release and inhibiting the reuptake of norepinephrine and dopamine, leading to a variety of excitatory neurologic responses. The dopaminergic stimulation may contribute to the sense of euphoria and the adrenergic effects to the heightened alertness that are hallmarks of the drug. The physiological and pharmacological study of methamphetamine has focused on the neuropsychological effects of the drug. However, the adrenergic stimulation also affects cardiovascular tissues and hemodynamic effects are readily seen at the doses commonly used in humans. Cardiovascular toxicity has been recognized for over 30 years and high catecholamine levels are known to be cardiotoxic, causing narrowing and spasm of the blood vessels, tachycardia, hypertension, stroke, and cardiomyocyte death. These findings are wellrecognized as acute effects of methamphetamine intoxication. In addition, chronic abuse at high levels of the drug addiction can also lead to a dilated cardiomyopathy, often of remarkable severity.
Cardiomyopathy is a common cause of death among long-term addicts and appears to be on the rise as the average duration of drug use increases (Herbeck et al. 2015). Presentation is gradual and insidious with typical symptoms of biventricular heart failure. Long-term studies of chronic methamphetamine abuse have suggested a strong relationship between duration of drug use and cardiomyopathy (Kaye et al. 2007). It appears that these individuals are also at risk for accelerated development of coronary artery disease. Ventricular dysfunction rarely improves upon discontinuation of the drug or the addition of medical therapies (Jacobs 1989;Lopez et al. 2009).
How this drug causes cardiomyopathy is not known, but more progress has been made in understanding the mechanism of neurotoxicity in chronic methamphetamine abuse (Tata and Yamamoto 2007). Such use produces long-lasting deficits in dopamine and serotonin nerve terminals with signs of axonal degeneration (Yamamoto et al. 2010). High doses of methamphetamine produce an increase in extracellular and intracellular dopamine that can rapidly auto-oxidize to form potentially toxic substances (Yamamoto et al. 2010) (Krasnova and Cadet 2009). It is this oxidation, as well as elevated levels of glutamate and mitochondrial dysfunction, that is thought to damage dopamine terminals and thus neurons (Tata and Yamamoto 2007;Krasnova and Cadet 2009).
Human studies demonstrate that males have greater dopamine release than women in response to methamphetamine, and may exhibit an increase in the degree of toxicity, suggested by a higher incidence of emergency department-related deaths involving meth (Dluzen and Liu 2008). In rodent studies, male mice also display greater meth-induced neurotoxicity than females (Wagner et al. 1993;Bourque et al. 2011).
These differences may result from estrogen. It has been reported that exogenous estrogen preserves striatal dopamine concentrations in gonadectomized female mice following meth administration (Dluzen and McDermott 2000). A similar neuroprotective response has been seen in estrogen-treated intact female mice (Dluzen and McDermott 2002). This neuroprotective effect occurs quickly and may be related to estrogen inhibiting meth uptake in the brain (Dluzen and McDermott 2006). Conversely, no neuroprotection by estrogen against meth was evident in either intact or gonadectomized male mice, suggesting that testosterone may increase neurotoxicity (Dluzen and McDermott 2006).
Compared to the brain, there has been little investigation of methamphetamine toxicity in the heart. It has been recognized that high doses of meth can cause vasoconstriction, myocardial infarction and fibrosis (Yu et al. 2002). In addition, meth directly depresses contractile function, intracellular Ca +2 handling and adrenergic response in isolated cardiomyocytes (Turdi et al. 2009). These early studies do not explore the long-term effects of meth exposure. Since human cardiomyopathy appears to develop only after long-term use at high dose, we examined the cardiac effects at 2 or 5 months of meth exposure in female and male mice. We took this opportunity to identify dysregulation of cardiac mRNA that may contribute to, or reflect, cardiac meth toxicity.
Estrogen appears to have a protective effect on the brain of meth-treated female mice and there are recent clinical observations that men are more susceptible to cardiac damage than women from meth exposure. Therefore, we examined differences in the response of male and female mice to long-term methamphetamine use by measurements of heart function, cardiac histology, and survival.

Materials and Methods
Animal treatments C57BL/6 mice were 8-to 10-week old at the beginning of this study. Mice were provided standard chow and water ad libitum and housed under a 12-hr light/dark cycle. They were killed by CO 2 asphyxiation for tissue extraction and histological analysis after 8 and 20 weeks. Intraperitoneal methamphetamine injections were performed weekly into the left upper quadrant of the abdomen using a 27 g needle. (+) Methamphetamine hydrochloride was purchased from Sigma Aldrich (St. Louis, MO), and dissolved in sterile phosphate-buffered saline (PBS). The dose was gradually increased 4-5 mg per week for 8 weeks for the 2-month study, reaching a final dose of 35 mg/kg, or 2 mg per week over 20 weeks to achieve a final dose of 40 mg/kg for the 5-month study. Control mice were injected with PBS using the same schedule. The 2-month study was conducted on C57BL/6 female and male mice once, whereas two distinct 5-month trials were performed on males and one on females. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Hawaii.

Echocardiography
Echocardiograms were performed on unanesthetized mice. The chest area was shaved with an electric razor and depilated with Nair â (Church and Dwight Co, Ewing, NJ). Echocardiograms were performed using the Vevo 2100 high-resolution imaging system (Visual Sonics, Toronto, Ontario) using a 38 MHz transducer. Mice were acclimated to handling during the days prior to examination. Left ventricular parasternal short-axis views were obtained from 2D B-mode and M-mode images just above the papillary muscles. Diastolic and systolic measurements were taken from M-mode images containing three consecutive beats. These measurements included left ventricular internal diameter diastole and systole (LVID;s, LVID;d), ejection fraction (EF), and fractional shortening (FS). Data from these measurements were analyzed using VisualSonics software.

Real-time PCR
RNA was extracted from homogenized heart tissue using TriReagent â (Molecular Research Center, Cincinnati, OH) following the manufacture's protocol. The RNA was further purified using an RNeasy â Mini Kit (Qiagen, Hildon, Germany), quantified on the Qubit fluorometer (Invitrogen, Carlsbad, CA) and examined on a 1% agarose-EthBr gel to confirm integrity. Single-stranded cDNA was synthesized from 1 lg of total RNA using QScript (Quanta Biosciences, Gaithersburg, MD). The mRNA levels of selected dysregulated genes from the microarray results were evaluated by semiquantitative RT-PCR with Roche Fast Start Universal SYBR Green (Mannheim, Germany) on a 7900HT Fast Real-time PCR system from Applied Biosystems (Foster City, CA).

Microarrays
After isolation and purification of RNA, Cy3 labeled nucleotides were incorporated using the Agilent low input quickamp kit (Santa Clara, CA). Individual control and methamphetamine samples from each trial were subjected to first and second strand synthesis followed by in vitro transcription to incorporate the dye into the new strand. After cleanup using the RNeasy â Mini Kit (Qiagen, Hildon, Germany), the samples were hybridized onto Agilent arrays (Mouse GE 4x44K v1) following the manufacturers protocol and scanned using the Agilent G2565CA Scanner. Intensities were extracted and data analyzed using Feature Extraction and Genespring Software (Agilent, Santa Clara, CA).

Histology
Hearts were perfused by cardiac puncture with PBS followed by 10% formalin in situ. Organs were subsequently fixed in 10% formalin at 4°C overnight and stored in 70% ethanol until embedding. Five-micron heart sections were deparaffinized and stained with picrosirius red (0.1% sirius red in picric acid). Sections were washed and dehydrated before imaging and analysis. Review and photography were carried out on the Zeiss Axioskop2 photomicroscope with bright-field illumination and an Olympus CCD color camera interfaced with a Scion CG-7 frame-digitizer-equipped computer. Images were captured using Image J v1.23 acquisition and analysis software (Scion Corporation & NIH).

Statistics
All results were expressed as mean AE SEM. Statistical significance was evaluated using two-tailed paired t-test between control-and methamphetamine-treated mice. A value of P < 0.05 was considered to be significant for all analyses.

Survival
C57Bl/6 mice, 8-to 10-week old, were treated weekly with increasing doses of methamphetamine or PBS by I.P. injections for 2 and 5 months. We saw no deaths in control or methamphetamine-treated animals after 2 months of progressively increasing treatment. In contrast, 5month methamphetamine treatment for both sexes ( Fig. 1) showed substantial mortality, confined only to males. Fewer than 50% of males survived the 5-month methamphetamine treatment, whereas no deaths occurred among the females.
Heart to body weight ratios (HW to BW) No significant difference between HW to BW ratios in female control (5.16 AE 0.17mgs/g) and methamphetamine (5.08 AE 0.09 mgs/g) treated mice were found in the 2month trial. Male-treated mice (5.89 AE 0.42 mgs/gram) exhibited a much larger change in HW to BW than their controls (4.98 AE 0.22 mgs/grams) ( Fig. 2A).
In the 5-month analysis, HW to BW ratios showed a small increase in treated females (PBS = 5.00 AE 0.60 mgs/ gram and meth = 5.53 AE 0.12 mgs/gram) but this did not reach statistical significance. A larger difference that did reach statistical significance was seen in male mice (PBS = 5.23 AE 0.10 mgs/gram and meth = 5.93 AE 0.19 mgs/gram) (Fig. 2B).
Based on the survival rate and echo data from the single 2-month study and the first 5-month trial, we felt a larger pool of methamphetamine-treated male mice was needed to confirm our findings. Hence, a second 5month trial was performed only on males, confirming the cardiac hypertrophy in these mice. No significant weight loss was observed for female or male controls, nor for treated animals during the 2-and 5-month study period (data not shown).

2-month echocardiography analyses
Cardiac function was evaluated by unsedated echocardiography three times during each trial. Baseline and 1month echo measurements were not significantly different between controls and treated animals. Therefore, only final values are shown in Table 1. At 2 months, there was a trend toward reduced FS and increased LVID in both male and female mice but only the decrease in FS in males reached statistical significance.

5-month female and male echocardiography analyses
Since we saw only modest changes after our 2-month regimen, and the human pathology is usually seen only after prolonged use, we repeated the study for 5 months with a gradual titration of methamphetamine to a higher final dose. Measurements were taken at baseline, two and a half months and then again at 5 months. Females treated for 5 months exhibited a small but not statistically significant decrement in cardiac function compared to controls (Fig. 3A-D). There was a small decrease in EF and FS between female controls (91.7 AE 0.65%, 61.8 AE 1.2%) and meth-treated animals (86.6 AE 1.6%, 54.9 AE 2.0%) at 5 months that did not reach statistical significance. The only statistically significant change in ventricular dimension occurred at the two and a half month time point where there was a significant increase between controls (2.6 AE 0.2 mm) and treated females (3.1 AE 0.1 mm) for the end diastolic internal diameter.
In contrast, males had substantial functional and structural changes in the heart (Fig. 4A-D). EF and FS measurements were significantly different between PBS A B Figure 2. Average HW to BW ratios of PBS-and Meth-treated mice. (A) 2-month trial showed significant difference between female PBS (n = 3) and Meth (n = 5) treated mice. Conversely, Meth-treated males (n = 6) had a greater average HW to BW ratio than PBS-treated controls (n = 3). (B) In the 5-month trial, a small but not statistically significant difference was found between Methtreated females (n = 7) and PBS controls (n = 4). The HW to BW ratio in Meth (n = 16) treated males was substantially higher than in males treated with PBS (n = 6).

Histology
Tissue sections of PBS and methamphetamine-treated hearts are shown in Figure 5A-D. A qualitative measurement was performed to examine collagen deposition in isolated heart sections using picrosirius red. Picrosirius stains collagen fibers red; the intensity and distribution of staining reflects the level of tissue fibrosis. In panels A and B, female 5-month control and treated heart sections show little to no fibrosis. In panels C and D there is a marked difference in male mice between the methamphetamine-treated tissue as compared to the control, revealing substantially more fibrosis and damage. No change was observed between the PBS and treated tissue from the 2-month study (data not shown).

Microarray data and real-time confirmation
A comparison of array and real-time expression data from a list of highly dysregulated transcripts after 5 months of meth treatment is shown in Table 2 and graphically in Figure 6. The most highly dysregulated transcripts after meth treatment in female mice were Bpifa1/Plunc, Reg3g and Cyp26b1. The most highly dysregulated transcripts in our male mice were Bpifa1/Plunc, Cyp2a5, Scgb1a1 and Arntl. On our microarrays, Bpifa1/Plunc expression was 3.7-fold (12.1-fold, 3.3-fold) higher in males as compared to females at 5 months. RegIIIg (Reg-IIIc) was also more abundant in our females and males showing a 3.4-fold increase in females and an 8.8-fold increase in males. Cyp26b1 was downregulated in females by -2.77-fold at 5 months and Arntl was downregulated in males by -2.40-fold for the same time period. Real-time semiquantitative evaluation confirmed these results. All dysregulated transcripts from male mice displayed the same direction of regulation as the microarray data. Transcripts from female mice, which showed lower overall changes in abundance, were confirmed by real-time PCR only about 70% of the time. Expression levels between females and males listed above were distinct. Males exhibited much higher upregulation than females for Bpifa1/ Plunc, Scgb3a2, Scgb3a1 and Reg3g, whereas females showed more downregulation for Cyp26b1 than males.  Highly dysregulated transcripts from 5-month Meth-treated mouse model. Only transcripts from known genes that were upregulated by >3.0fold or downregulated by <-2.0-fold are listed above. Data are presented on a log 2 scale. P value <0.05 for all microarray values. Data represent the average result from three individual microarrays from three separate mice. All results have been submitted to GEO under accession # GSE109790.

Discussion
We describe functional, structural and molecular differences between female and male mice treated with methamphetamine for 5 months. This model demonstrates the cardiac toxicity and dilated cardiomyopathy also seen in human addicts and recapitulates the clinical observation of greater male susceptibility. Very little change was observed in the echocardiography portion of our 2-month methamphetamine trial in both sexes and in our histology results. Little functional or histological change was observed at 2 months in either sex. The sexual dimorphism in survival was striking with 56% mortality among males, mostly in the last month of treatment, and none in females. There is an interesting corollary to this finding in human clinical observations where ER visits for chest pain are much higher among male than female meth addicts and decedents show a large male predominance (Karch et al. 1999;Turnipseed et al. 2003;Ito et al. 2009;Herbeck et al. 2015).
We performed an in vivo assessment of the heart using echocardiography and observed a decrease in cardiac function in males after 5 months of progressively increasing methamphetamine injections. In our 2-month study, our results reflect an earlier stage in the myopathic process. Although there was a decrease in fractional shortening between treated and control males, as well as a significant increase in the average male HW to BW ratio, we did not detect any mortality or myocardial fibrosis in males or females.
In our 5-month study, in addition to the marked mortality, we saw a substantial increase in HW to BW ratio, significant decrement in cardiac function and a remarkable increase in the amount of cardiac fibrosis in male compared to female mice. It is evident that dilated cardiomyopathy develops more slowly in methamphetamine-treated female mice than males. Although not amenable to statistical evaluation, the fibrosis seen in the single surviving male illustrates another important detrimental effect of methamphetamine exposure, which likely contributes to the functional and gravimetric effects that we observed. It is possible that we could be under-estimating the effect of meth on cardiac function in the male mice, as the mice that died may have had worse function than those that lived, (although we cannot prove this).
To further evaluate the underlying molecular basis for the changes seen in our model we performed transcriptional analysis on the hearts of treated and control mice. We identified several dysregulated transcripts involved in inflammation, metabolic processes and circadian rhythm. Bpifa/Plunc was the most dysregulated transcript. It is thought to have antimicrobial as well as anti-inflammatory functions in the trachea but is not commonly encountered in the heart, although we have previously found it to be upregulated in a model of chronic doxorubicin toxicity and downregulated in a cardiomyocyte-specific endothelin-1 knockout (Yi et al. 2006;Bingle and Bingle 2011;Gally et al. 2011;Lukinskiene et al. 2011). Other transcripts related to inflammation were also more abundant in methamphetamine exposed hearts. RegIIIg, Scgb3a2 and IL6 were substantially upregulated in both females and males with the exception of IL6 which was only slightly upregulated in meth-treated males. IL6, a cytokine produced by inflammatory cells, is responsible for regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs (Banerjee et al. 2009;Rincon and Irvin 2012). It has pleotropic effects in the heart but is generally thought to be profibrotic (Banerjee et al. 2009). It is thus unclear why IL6 transcript was more abundant in our female mice, however, the function of this cytokine depends on the abundance of its receptor and other aspects of the local milieu, which could be modified by estrogen.
The transcripts for RegIIIg and Scgb3a2 were more abundant in our treated male mice than our females. RegIIIg is known to play a role in innate immune functions and bacterial colonization of the gut (Wu and Chakravarti 2007;Johansson and Hansson 2011;Matsumoto et al. 2012). Like RegIIIg, Scgb3a2 is predominantly expressed in the pulmonary airway epithelium and plays a role in lung inflammation (Chiba et al. 2006;Lu et al. 2011). The marked upregulation of Bpifa/Plunc, RegIIIg and Scgb3a2 suggests a surprising role for immune mediators not previously appreciated to have important roles in methamphetamine cardiomyopathy.
In summary, we have developed a chronic mouse model of meth-induced cardiomyopathy that is similar to the clinical presentation seen in human patients. We have determined that long-term meth exposure increases HW to BW ratios in both male and female mouse hearts while substantially reducing the survival rate of our males. It appears that this long-term exposure is important to obtain and evaluate cardiac pathology, especially as these changes were not seen with a shorter, 2-month, treatment schedule.
Additionally, with the long-term protocol that we used, meth treatment contributes to the development of cardiomyopathy in male mice with less effect on females. We have identified several highly dysregulated transcripts, Bpifa/Plunc, IL6, RegIIIg, and Cyp26b1, that may play a role in heart protection or injury and may do this by regulating important pathways involved in inflammation or toxicity. It is interesting that we find a gender discordance in rates of death, HW to BW ratios, cardiac dilation, and histological evidence of fibrosis. Although the hearts of treated female mice do show some evidence of dilation and dysfunction, death, perhaps due to dysrhythmia, is disproportionately found in the males. One possible explanation could be that estrogen has a greater protective role in the heart during the course of drug use than previously thought.
Another possibility is that females metabolize methamphetamine more efficiently and/or with less toxicity and that this pharmacokinetic effect protects the female heart. It will be important to determine how estrogen, or female gender, affects meth metabolism in long-term users. Future studies in rodents may help to elucidate how this popular and devastating drug selectively damages the heart in men.


extfieldsofstudy: Biology Medicine

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Pattern Recognition by Melanoma Differentiation-Associated Gene 5 (Mda5) in Teleost Fish: A Review

Teleost fish, as with other vertebrates, rely on their innate immune system as a first line of defense against invading pathogens. A very important characteristic of the innate immune response is its ability to recognize conserved molecular structures, such as viral dsRNA and ssRNA. Mda5 is one of the three pattern recognition receptors (PRRs) that recognize cytoplasmic viral ligands. Teleost Mda5 is widely conserved among several fish species and possesses the same structural domains as those seen in their mammalian counterparts. Fish Mda5 has been shown to be capable of initiating an inflammatory response both in vitro (in different fish cell lines) and in vivo using synthetic viral analogs or virus. The interferon (IFN) pathway is triggered as a result of Mda5 activation, leading to the expression of type I IFNs, IFN- stimulated genes and pro-inflammatory cytokines. Although it is known that Mda5 acts as a receptor for virally-produced ligands, it has been shown more recently that it can also initiate an immune response against bacterial challenges. This review discusses recent advances in the characterization of teleost Mda5 and its potential role in antiviral and antibacterial immunity in teleost fish.

INTRODUCTION
Vertebrates have both innate and adaptive immune systems that help them defend themselves against pathogens, such as viruses and bacteria. The innate immune system acts as the initial line of defense against infection and plays a pivotal role in mediating an immediate immune response, which in turn helps to activate the adaptive immune system (1). While higher vertebrates (i.e., mammals), have a much more complex adaptive immune system compared to lower vertebrates, studies have shown that lower vertebrates (including fish) have an intricate innate immune system that compensates for their less developed adaptive immune system (2).
Early pathogen recognition is paramount for an organism's survival. It is important that the host has a set of "sensors" that can instantly recognize the presence of microbial/viral nucleic acids within its cytoplasm. One of these is the DExD/H-box (DDX) protein family that includes RNA and DNA helicases possessing a DExD/H-box domain. DDX proteins are directly involved in the regulation of gene induction and other important processes including signal transduction, gene promoter regulation, mRNA splicing, translational regulation and most importantly, they have been implicated in innate immunity, acting as RNA sensors or signaling molecules (3). Another group of receptors that recognize the presence of cytoplasmic nucleic acids are the pattern recognition receptors (PRRs), which are more thoroughly studied and well-characterized in most vertebrates. PRRs are innate immunity receptors, defined by their ability to specifically recognize microbes and/or microbial moieties (4). In the presence of invading pathogens, the innate immune response is initiated primarily through the recognition of conserved pathogen-associated molecular patterns (PAMPs) by the PRRs (5). These PRRs serve as a pathogen surveillance system in all eukaryotic organisms, which recognize the conserved molecular pathogen signatures comprised of proteins, lipids and nucleotides (6). The PRRs, therefore, allow the immune system to distinguish self from non-self, while still retaining the capacity to respond effectively during an infection. Ultimately, the recognition of PAMPs by the PRRs trigger the activation of multiple signaling cascades in the host immune cells, including the stimulation of interferons (IFNs) and several other cytokines (6,7).
PRRs are categorized into three groups depending on their function: (i) soluble bridging PRRs, which facilitate the recognition and elimination of their ligands by phagocytes, (ii) endocytic PRRs, which mediate the recognition and internalization of microbes and/or microbial moieties, and (iii) signaling PRRs, which are involved in cell activation in response to a diverse range of microbial moieties (4,8). Signaling PRRs are functionally very distinct from the other groups of PRR and are further sub-categorized into three different groups, namely: (i) toll-like receptors (TLRs); (ii) nucleotide oligomerization domain-like receptors (NLRs) and (iii) retinoic acid-inducible gene-I (RIG-1)-like receptors (RLRs) (7, [9][10][11][12]. RLRs belong to DExD/H box RNA helicases that are known to be the core cytosolic receptors involved in the recognition of viral RNAs. In mammals, three members in the RLR family have been observed, retinoic acid-inducible gene 1 (RIG-1 or DEAD box polypeptide 58, DDX58), melanoma differentiation-associated gene 5 (Mda5, interferon induced with helicase domain 1, IFIH1, or Helicard), and laboratory of genetics and physiology 2 (LGP2 or DExH box polypeptide 58, DHX58) (13) The RLRs observed in mammals are found to be conservatively present in teleost fish. In fact, all three members, RIG-1, Mda5 and LGP2, have been identified in a range of fish species (14).
The recent advances made in the field of fish immunology over the past few decades, specifically on the knowledge of RLRs in teleost fish, has led the way for to a better understanding of the fish immune system, as well as the diversity and evolution of antiviral immunity in vertebrates. Thus, in this review, we focus on the recent discoveries in relation to PRRs, focusing on Mda5 in particular, which had been identified in several fish species, including model fish species such as zebrafish (Danio rerio), and some economically important fish species such as rainbow trout (Oncorhynchus mykiss) and Japanese flounder (Paralichthys olivaceus).

GENERAL STRUCTURE OF MDA5 AND ITS ORTHOLOGS IN FISH
Mda5, together with RIG-1, are cytoplasmic sensors of dsRNA comprising of four discrete domains; two caspase recruitment domains (CARDs) at the N-terminal region, a DEAD/DEAH box helicase domain (DEXDc), a regulatory domain (RD) and a helicase C-terminal domain (HELICc) (15,16) as shown in Figures 1A,B. As observed in humans, Mda5 and RIG-1 proteins in fish are closely related proteins, having structural similarities of 23 and 35% amino acid (aa) identity in their N-terminal tandem CARD and C-terminal helicase domains, respectively (17).
Mda5 (and RIG-1) in fish consists of protein domains that are similar to their mammalian counterparts. The first fish ortholog of Mda5 was reported in pufferfish (Fugu rubripes) in 2008, through the use of bioinformatic analyses of available whole genome sequences (18). Since the first characterization of Mda5 in fish, studies focusing on the analysis of this important PRR have increased significantly, partly because of the recent advances made in bioinformatics.
The Mda5 gene has now been cloned and characterized for a number of fish species. Differences in the aa length is quite noticeable when comparing the cloned Mda5 among fish species. As shown in Figures 1A,B, this difference can be attributed to the regions of low compositional complexity along the whole sequence (represented as pink boxes in the diagram). To further elucidate this, the six essential domains of Mda5 were analyzed through the use of the Simple Modular Architecture Research Tool (SMART). As predicted by the tool, the domains were found at different position along the Mda5 sequence in different teleost species, moreover, the aa length of the respective domains also differ ( Table 1). These subtle differences in the sequence of Mda5 between different teleost species does not appear to interfere with the function of the protein. Though they differ in ORF length and number of aa residues (see Table 2), analysis of their protein residues show that there is a close phylogenetic relationship between the Mda5 from different fish species and they all have a significant similarity with other vertebrate Mda5 ( Figure 1C).

MDA5 AND ITS INVOLVEMENT IN THE INNATE IMMUNE RESPONSE
In 2002, Mda5 was initially discovered as an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties in human melanoma cells (19), and then in 2004, Mda5 was reported to play a major role in an intracellular signal transduction pathway, resulting in the activation of the IFNβ promoter, and V proteins of paramyxoviruses were shown to interact with Mda5 to block its activity (20). Subsequent studies have indicated that Mda5 is capable of recognizing a viral infection and transmitting a signal by CARD (21,22). It was also established that Mda5 could sense single stranded RNA with 5' triphosphate and could selectively recognize long dsRNAs (>3 kb) (18,23), which includes dsRNA replication intermediates of positive-sense RNA viruses, the genome of dsRNA viruses and polyinosinic: polycytidylic acid (poly I:C). The ability of Mda5 to recognize these dsRNA-related molecules induces the secretion of type I IFN, in particular (24). There had been underlying issues regarding the specificity of ligands that Mda5 and RIG-1 recognized, suggesting an overlap in the mechanism of action between these two RLRs. RIG-1 can be activated by diverse positive-and negative-strand RNA viruses including influenza, Rift Valley fever, measles, Ebola, vesicular stomatitis virus (VSV), and hepatitis C viruses (25). The minimal requirement to activate RIG-1 is a blunt-ended base-paired RNA 10-20 bp long with a 5' triphosphate and free mismatches near the blunt end (denoted 5' ppp bpRNA, since this could arise from ssRNA with complementary ends or dsRNA) (26)(27)(28). It is also reported that much longer dsRNAs (>200 bp), including poly (I:C), which does not necessarily bear a 5'ppp-end or blunt-ended, can also induce IFN via RIG-1 (23). Thus, it is noteworthy to mention that although poly (I:C) (a synthetic dsRNA commonly used to represent a viral ligand in Mda5 and RIG-1 studies) can induce IFN production through Mda5 and RIG-1 activation, these two RLRs are capable of distinguishing this ligand according to the size wherein, Mda5 tends to recognize long poly (I:C) and RIG-1 specifically reacts with short poly (I:C). Although the mechanism of how these RLRs discriminate between differences in length is not yet understood, we think that it has to do with the "uncoiling" of the dsRNAs and how much ATPase activity is involved for the Mda5 or RIG-1 to be activated.
The CARD and helicase domains of the Mda5 are the domains directly involved in the initiation of the signaling pathway, and triggering the innate immune response. The helicase domain binds to dsRNA leading to the activation of the CARD domains. After interacting with PAMPs, the Mda5 CARDs are exposed and they form a complex with the CARD domain of the mitochondrial protein IFN-promoter stimulator-1 [IPS-1, also known as mitochondrial antiviral signaling (MAVS), virus-induced signaling adapter (VISA), and CARDIF], which is located on the outer membrane of the mitochondria (39)(40)(41)(42). This is then followed by the recruitment of tumor necrosis factor (TNF)-receptor associated factor 3 (TRAF3) and activation of TRAF family member-associated NF-κB-activator binding kinase-1 (TBK1) and inducible IκB kinase (IKKǫ) (40,41,43). The activation of these kinases result in the phosphorylation of interferon regulatory factor 3 and 7 (IRF3/7), the phosphorylated IRF3/7 forms a dimer and translocates to the nucleus to activate the type-I IFN promoter (44). Ultimately, these processes initiate  the expression of IFN and pro-inflammatory cytokines. The expression of IFN triggers the release of antiviral effectors, such as IFN-stimulated gene (isg) 15, myxovirus resistance gene (mx), 2' , 5'-oligoadenylate synthetase (OAS)-directed ribonuclease L (RNASEL) pathway and protein kinase R (PKR), which in turn enhances the IFN-mediated antiviral response (45) (Figure 2).

MDA5 IN VIVO AND IN VITRO EXPRESSION IN TELEOST
Synthetic RNAs, IFNs and viruses are known to induce the expression of Mda5 in mammals (21,46). Studies have shown that fish Mda5 is also capable of responding, both in vivo or in vitro, to stimulation by synthetic double-stranded RNA (dsRNA), poly(I:C) (30,32,35) and to viral infections (29,33,34). The CiMda5 transcripts in grass carp (Ctenopharyngodon idella) have been observed to increase in expression after infection with grass carp reovirus (GCRV) in vivo, especially in the spleen and liver (29). When expression of rainbow trout Mda5 was examined in vitro using rainbow trout gonad (RTG-2) and spleen (RTS-11) cell lines after stimulating the cells with poly(I:C), Mda5 transcripts were observed to increase in both cell lines, but this stimulation was greater in the RTS-11 cells. Intracellular poly(I:C) stimulation also caused a significant increase in Mda5 expression. The expression of Mda5 in RTG-2 cells could also be stimulated using synthesized IFNs (16). Expression of Japanese flounder Mda5 was evaluated in vitro using whole kidney leukocytes (KL) and peripheral blood leukocytes (PBL) with poly(I:C) stimulation, and also in vivo with viral hemorrhagic septicemia virus (VHSV), with significant up-regulation of Mda5 transcripts noted both in vitro and in vivo (30).
The studies outlined above have clearly shown that Mda5 is able to be stimulated, both appropriately and efficiently, by synthetic stimulators such as poly(I:C) and viral infections in either fish or in fish cell lines, and have laid the ground work for subsequent studies examining the Mda5 response to viral infections in other fish species. As summarized in Table 3, fish Mda5 can strongly be up-regulated in different teleost species through the use of different stimulants, either with a virus or poly(I:C) as observed in different cell lines and organs.
Mammalian Mda5 is established as a viral PAMP-recognizing PRR of different ssRNA, dsRNA viruses as well as poly(I:C), which is a synthetic analog of dsRNA virus. In the case of teleost Mda5, this PRR has been implicated in the stimulation of the immune response against viral antigens, probably by serving as a sensor. However, in the study performed by Ohtani et al. (30), in which they used a synthetic bacterial analog, Lipopolysaccharide (LPS), representing stimulation by Gram-negative bacteria, their results showed up-regulated Mda5 expression after LPS stimulation, suggesting that Mda5 might not be involved exclusively in recognizing viral PAMPS, but they are also capable of indirectly distinguishing bacterial PAMPs. Several studies concurrently showed that LPS or bacterial challenge resulted in up-regulation of fish Mda5, such as in channel catfish (Ictalurus punctatus) challenged with Edwardsiella ictaluri (31), in common carp (Cyprinus carpio) after Aeromonas hydrophila challenge (2) and in black carp (Mylopharyngodon piceus) MPF cells after LPS stimulation (36).
The most recent studies on fish Mda5 further verify that this PRR is indeed not only involved in virus detection, but also has the ability to initiate the RIG-1/Mda5 pathway during bacterial infection. The expression level of Asian seabass Mda5, AsMda5, in response to bacteria was elucidated by infecting juvenile fish with either Vibrio alginolyticus (Gram-negative bacterium) or Staphylococcus aureus (Gram-positive). When Sahul India seabass kidney (SISK) cell line was exposed to LPS, a sustained level of AsMda5 up-regulation was obtained several hours after stimulation, but the levels of expression obtained were not as high as those seen in fish stimulated with LPS in vivo. Poly(I:C)injected fish, on the other hand, produced much higher levels of AsMda5 expression than fish injected with bacterial LPS (37). In tilapia (Oreochromis niloticus), a Streptococcus agalactiae (Grampositive bacterium) infection caused an increase in OnMda5 transcript levels in the intestine, kidney, gills and blood at different time points (38). Together, the results highlighted above for the various fish species indicate that fish Mda5 is not only involved in antiviral immune responses, but also bacterialtriggered immune responses, although the mode of action of Mda5 stimulation by bacteria has yet to be determined.
Bacterial ligands are recognized by a different group of PRRs, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and some toll-like receptors (TLRs), which had been widely observed among vertebrates. NLRs can cooperate with TLRs and regulate inflammatory and apoptotic responses. Different mammalian TLR families had been elucidated and most of them have also been found in teleost fish including two additional fish-specific TLR family members (47,48). Although these fish orthologs have already been demonstrated in different species, the role of these TLRs in the recognition of ligands from bacteria is now the focus of intense studies. NLRs, specifically, NOD1 and NOD2, recognize peptidoglycan components common to both Gram-positive and Gram-negative bacteria. Both proteins drive activation of mitogen-activated protein kinase (MAPK) and nuclear factor κ-light chainenhancer of activated B cells (NF-κB) pathways, leading to proinflammatory cytokine production (49,50). There has not been a direct link between these receptors, except for the fact that when these receptors are activated, adaptor proteins (i.e., MyD88, MAVS) trigger a downstream cascade leading to the release of inflammatory genes needed in the fight against pathogens (51). The studies mentioned above, on the overexpression of Mda5 after bacterial challenge, do not provide any additional information on the mechanism involved or if it is definite that Mda5 recognized these bacterial ligands. We, therefore, hypothesize that maybe the presence of excess number of bacterial nucleic acids (i.e., small RNAs) which were indirectly sensed by Mda5 led to the observed overexpression of this PRR. We also believe that if they had included RIG-1 in their analysis, an up-regulation of RIG-1 could also have been observed, but the study focused more on Mda5, and they did not delve into TLR/NLR markers. The limiting factor of previous studies is that lack of expression of any TLR/NLR as a comparison, as this would further explain if these two totally different group of PRRs perform overlapping immune response mechanisms in fish.

ANTIVIRAL AND ANTIBACTERIAL FUNCTIONS OF MDA5
The essential role of Mda5 in antiviral responses had been suggested by the existence of paramyxovirus proteins. The highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds to Mda5 products. As shown from reporter assays, Mda5 stimulates the basal activity of the IFN-β promoter, and over-expression of Mda5 enhances the activation of IFN-β in response to intracellular dsRNA. It was also shown that Mda5 can activate both NF-κB and IRF-3, suggesting that Mda5 plays a pivotal role in the upstream activation of these transcription factors in response to dsRNA, however, these activities were repressed by co-expression of the V proteins (20). The V protein of the Sendai virus, Hendra virus, simian virus 5, human parainfluenza virus 2, and mumps virus selectively abrogates Mda5 function, highlighting the ingenious mechanisms of initiating antiviral immune responses and the action of virus-encoded inhibitors (17,20).
It was demonstrated using knockout mice that Mda5 plays a crucial role in type I IFN responses by dendritic cells (DCs) and macrophages, when the mice were stimulated with poly(I:C). Specifically, Mda5-deficient mice showed abrogated production of IFN-α and IFN-β in bone marrow-derived DCs and macrophages, and that this PRR is functionally dominant over TLR3 for type I IFN responses to poly(I:C) in vitro and in vivo. Furthermore, mice without Mda5 activity succumbed sooner to infection by encephalomyocarditis virus (ECMV), confirming the essential role that Mda5 has in the host's resistance to ECMV in vivo (21).
In comparison to mammals, innate immunity in teleost fish is poorly understood. It is therefore important to establish how PAMPs recognize PRRs in fish. Earlier reports on fish PRRs, especially Mda5, focused on molecular characterization instead of function, and it is only in the past decade that studies have focused on the role of these molecules in the innate immunity of fish. When the effect of grass carp Mda5 (CiMda5) on the production of various IFNs in C. idella kidney (CIK) cells infected with grass carp reovirus (GCRV) was examined, CiMda5 was shown to induce an extensive IFN response in the infected cells by facilitating total phosphorylation of IRF3 and IRF7, enhancing the heterodimerization of IRF3 and IRF7 and the homodimerization of IRF7. The homodimer IRF7 broadly induces the production of IFN-1 in response to GCRV infection, suggesting that CiMda5 has a crucial role in the cytosolic pathway for the induction of IFN genes in response to GCRV (52). Meanwhile, over-expression of Mda5 in Hirame natural embryo (HINAE) cells resulted in a decreased cytopathic effect in cells infected with VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV). The observed reduction in VHSV titers indicate that Japanese flounder Mda5 inhibits the replication of ssRNA viruses (VHSV and HIRRV) as well as dsRNA viruses (IPNV). Moreover, the expression level of type I IFN, Mx and ISG15 genes in Mda5-overexpressing HINAE cells, infected with VHSV, was significantly higher than in noninfected cells. These results demonstrate the ability of Japanese flounder Mda5 to enhance the antiviral activity of the fish, mediated by the activation of type I IFN and IFN-stimulated genes (30). In the zebrafish cell line ZF4, overexpression of the two splice variants of Mda5 (Mda5a and Mda5b) significantly induced type I interferon promoter activity and promoted protection against SVCV infection in transfected cells (5). In another zebrafish experiment, overexpression of Mda5 in zebrafish liver (ZFL) cells had a 2.5 × 10 6 -fold reduction in viral burden after infected with snakehead rhabdovirus (SHRV) demonstrating that Mda5 overexpression increases resistance to SHRV (1). Overexpression of orange spotted grouper (Epinephelus coioides) Mda5 triggered an increase in the expression levels in IFN and IFN-stimulated response element (ISRE) promoter in a dose-dependent manner (400 and 800 ng ml −1 ) and also enhanced the expression of IRF3, IRF7, and TRAF6 (TNF receptor-associated factor 6) and some proinflammatory factors including, tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-8 at different time points during SGIV and RGNNV infection (34).
The role of Mda5 in the fish innate immunity by the induction of IFN-mediated immune response after viral infection has been well-elucidated. In addition, studies have demonstrated that Mda5 can also be triggered by bacterial stimulation. However, the studies carried out on fish, showing the ability of bacterial stimulants in up-regulating Mda5 expression, did not specifically discuss the mechanism behind this occurrence, but it has also been observed in other animals. In fact, there are studies in mammals which show that the RIG-1/Mda5 pathway, thought primarily to detect viruses, is also involved in the innate immune response to intracellular bacteria e.g., Legionella pneumophila, a Gram-negative bacterium (53) and Listeria monocytogenes, a Gram-positive bacterium (54). Listeria monocytogenes releases nucleic acids during the infection that are recognized by the cytosolic sensors RIG-1, Mda5, and stimulator of interferon genes (STING), thus resulting in the expression of IFN-β and an inflammasome response (54).
The involvement of Mda5 in the innate immune response in fish is limited to the results presented above, and it has not been confirmed that Mda5 acts as a receptor for viral and/or bacterial ligands. Instead, we can only generalize that the overexpression of Mda5 can lead to the protection of fish, therefore, investigating the "ligand-receptor" interaction of fish Mda5 could give us a better insight if this PRR has an equivalent function to that observed in higher vertebrates.

INTERACTION OF MDA5 WITH LGP2
Our understanding of fish immunology had increased greatly over the past few decades, including the discovery of orthologous genes for mammalian RIG-1, Mda5, and LGP2 (14). The functional characteristics of these RLRs have been investigated in a range of teleost species, including model fish species such as zebrafish, and these genes appear to have similar functions to those present in mammals [5,53].
All three RLRs share homologous core structural domains, including a DExD/H box helicase domain, a helicase Cterminal domain and a C-terminal domain (CTD), however, while RIG-1 and Mda5 have CARDs, LGP2 lacks them (55), and this absence of CARDs in LGP2 makes it unable to induce signaling alone. This is consistent with the inability of LGP2 to intrinsically activate the IFN-β promoter in transient overexpression experiments (56). Thus, it is difficult to determine its exact role in RLR-mediated signaling (57), and until now, the function of LGP2 in antiviral signaling has been controversial.
LGP2 has been identified as a negative regulator of IFN response, for example when triggered by Sendai virus, Newcastle disease virus (17,56,58,59) or poly (I:C) (58). However, mice studies have shown that LGP2 can have either a positive or a negative role in IFN induction (60). The action of LGP2 is believed to synergize with that of Mda5, but not RIG-1, to boost IFN signaling at low levels of LGP2 expression. On the other hand, at higher levels of LGP2 expression, it acts as an inhibitor of RIG-1 and Mda5 signaling (61)(62)(63). In recent studies, mammalian LGP2 has been shown to be involved in Mda5 filament formation and Mda5-mediated viral RNA recognition (63,64). Other studies have confirmed that LGP2 synergizes with Mda5: (1) to elevate IFN-transcription in vivo, for example during an encephalomyocarditis virus infection or poly (I:C) stimulation (62); (2) to facilitate viral RNA recognition through its ATPase domain (65) and (3) to sense Sendai virus infection for IFN-1 induction along with the loss of RIG-1, as determined in Chinese tree shrew (66).
Some reports have shown that teleost LGP2 is a negative regulator of antiviral immunity when overexpressed in vitro. For example, overexpression of crucian carp (Carassius carassius) LGP2 reduced the activity of IFN promoters, mediated by RIG-1 and Mda5 (67), and down-regulation of antiviral immune genes like Mda5, was also observed in cells overexpressing the grouper LGP2 (68). In zebrafish, LGP2 negatively regulates the IFN response mediated by poly (I:C) by blocking some of the important signaling factors, including RIG-1 and Mda5, but not IRF3/7 (69). It also appears that the antithetical function of LGP2 in antiviral immunity depends on LGP2 expression levels, similar to that is observed with mammalian LGP2. Zebrafish LGP2 functions as a positive regulator of IFN signaling during the early phase of virus infection; during this time RIG-1 and Mda5 are expressed at low levels, while during latter phases of the infection, LGP2 adopts a negative role. However, the maximum stimulatory effect of zebrafish LGP2 is lower than levels of Mda5 and RIG-1 expression (70). These results have also been corroborated for grass carp LGP2. During the resting state and early phase of grass carp reovirus (GCRV) infection, synthesis and phosphorylation of IRF3/7, and mRNA levels and promoter activities of IFNs and NF-κBs are inhibited, at a time when grass carp LGP2 is overexpressed. Luciferase assay have shown that grass carp LGP2 binds to RIG-1 and Mda5 with diverse domain preference, and this binding is independent of the GCRV infection. Another interesting result showed that grass carp LGP2 inhibits K63-and K48-linked RIG-1 and Mda5 ubiquitination, resulting in suppression of protein degradation. These results indicate that LGP2 has a role as a suppressor in RLR signaling pathways, which is important in maintaining cellular homeostasis during the resting state and early phase of GCRV infections (71).
On the other hand, black carp LGP2 (bcLGP2) was clearly shown to have a synergistic effect with bcMda5 using reporter assays, in which, both the induction of zebrafish IFN3 and fathead minnow IFN (eIFN), mediated by bcMda5 and bcLGP2, were much higher than that obtained by bcMda5 alone, and was higher than the combined effect of bcMDa5 or bcLGP2 alone. The synergistic function between bcLGP2 and bcMda5 reflects bcLGP2 effect on the antiviral activity of the host. Epithelioma papulosum cyprini (EPC) cells, overexpressing both bcLGP2 and bcMda5, showed a decrease in CPE development and viral titer during infection with GCRV or SVCV, in contrast with cells expressing either bcMda5 or bcLGP2 alone (36). In a study focusing on rainbow trout Mda5 and LGP2, it was evident that both RLRs are capable of binding to poly (I:C), triggering IFN production. Also, Mda5 expression is not affected by the overexpression of LGP2 in transfected cells, and vice versa, implying that these RLRs function in parallel as positive regulators for IFN production (16). Although these results help to substantiate the synergy between LGP2 and Mda5, the mechanism behind their interaction remains unclear. A proposed mechanism of action showing the antithetical role of LGP2 with Mda5 is suggested in Figure 3.
Orthologs of Mda5 and LGP2 seem to be common to all teleost families, unlike RIG-1, which is only found in more primitive fish species, such as those in classes Ostariophysi, Protacanthopterygii, and Paracanthopterygii (77). It is believed that Mda5 might have emerged before RIG-1 and that the domain arrangements of the genes evolved independently by domain grafting rather than a simple gene duplication event (18).
Furthermore, the presence of the three RLRs in very ancient fish, Sarcopterygii, imply the loss of RIG-1 after the divergence of the Acanthopterygii from the Paracanthopterygii LGP2 in the RLR signaling pathway. The presence of dsRNA in the cytoplasm is recognized by both RLRs, however, the signaling mechanism can follow two different paths: (a) the LGP2 acts as a negative regulator of type I IFN response that causes inhibition of the Mda5 and suppression of the expression of MAVS, IRF3, and IRF7 and (b) the LGP2 functions as a positive regulator in which LGP2 nucleates MDA5 filaments by binding dsRNA termini extending the MDA5 filaments. The LGP2 stabilizes the formation of these shorter filaments for the proper attachment to MAVS, thereby, causing more efficient and up-regulated expression of IRF3 and IRF7, leading ultimately, to the up-regulation of secreted type I IFNs and ISGs. (77). Interestingly, most of the fish species reared for aquaculture do not possess the RIG-1 ortholog, and all of them belong to the superorder Acanthopterygii.
With the absence of RIG-1 in some of the Acanthopterygians, their RLRs have possibly evolved differently, wherein their Mda5 as well as LGP2 perform most of the pivotal role in antiviral sensing. Knowing that LGP2 and Mda5 have the capability to work synergistically, it is important to help establish if these two RLRs function in place of RIG-1 in these fish species, and if not, what is the equivalent gene that performs the role of RIG-1? For instance, in chicken, another organism that lacks RIG-1, studies found that chicken Mda5 compensates for the lack of RIG-1 by preferentially sensing shorter dsRNA synthetic poly (I:C) instead of long dsRNAs (78). Another example of mammal that does not have RIG-1 is the Chinese tree shrew. It was revealed that the loss of RIG-1 brought positive selection signals to tree shrew Mda5(tMda5) and LGP2(tLGP2). Data showed that tMda5 alone or tMda5/tLGP2 could replace RIG-1 as a sensor for RNA viruses that trigger IFN production (66). It is believed that this replacement is enhanced due to the interaction of tMda5 with tMITA (Mediator of IRF3 activation), which interacts with RIG-1 resulting in a cascade of antiviral signaling (79). This information tells us that even in the absence of RIG-1, the innate immune system has a way of compensating for the loss of some molecules by relying on other functional molecules, probably homologs, although in fish, the compensatory effect of Mda5 in teleost lacking RIG-1 has not yet been verified, and further investigation is essential to establish this. A study was performed on the RLRs, Mda5, and LGP2 of rainbow trout (Oncorhynchus mykiss), focusing on the parallel function of these two RLRs that synergistically increase in the production of IFNs (16), but whether this was in compensation for the lack of RIG-1 in this fish was not specifically discussed.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The teleost immune system may not be as elaborate as that of its mammalian counterparts, but they have an intricate innate immune system that is on a par with the complex immune system present in mammals. Since the aquatic environment in which fish live is very different to that of mammals, where they are in close contact with pathogens, it is important that their innate immune system offers a first line of defense against invading pathogens. The PRRs in teleosts, similar to that present in mammals, are capable of sensing pathogens and inducing antiviral and/or antibacterial responses. Knowing the role that Mda5 plays during an infection will help give a clearer insight of how the teleost immune system works. Mda5, together with other RLRs, are able to sense pathogens and, in turn, activate downstream processes in the fish's immune response, ultimately, preventing them from succumbing to the infection. Although, Mda5 was described for several different teleost species in this review, the mechanism of action of Mda5 still needs further elucidation. As discussed, Mda5 expression directs the recruitment of the downstream adaptor MAVS from the mitochondria, then associates with signaling molecules like TBKI, TRAF3, and MITA, which in turn facilitate the activation and phosphorylation of IRF3 and IRF7, leading to their translocation into the nucleus for the induction of type I IFNs and ISGs. These downstream molecules have been identified in various teleost species as a result of stimulation with poly(I:C) and LPS, as well as viral and bacterial infections, however, the extent to which Mda5 regulates the whole process of initiating innate immunity in fish has yet to be established and whether Mda5 works in cooperation with other PRRs thereby suggesting a network of immune molecules instead of a single, linear pathway.
Further studies are needed to establish how these PRRs function within the teleost immune system, for example: 1. RLRs that recognize viral ligands include RIG-1, Mda5, and LGP2. RIG-1 and Mda5 recognize distinct viral dsRNA.
LGP2, on the other hand, recognizes the same viral ligand as RIG-1. The molecular signaling mechanisms of RIG-1 and Mda5 are known to share some common features and LGP2 has been found to be a co-stimulatory molecule for Mda5. It appears that these RLRs have overlapping mechanism of action upon virus invasion. It is therefore important to further characterize their function to be able to differentiate them from one another, especially with respect to the downstream signaling cascade they initiate during an antiviral response. 2. Mda5 shows positive up-regulation in the presence of bacterial ligands, such as LPS and bacteria (both Gram positive and negative strains) suggesting that its activity is not limited only to viral, but also bacterial sensing. Since most of the studies relating to teleost Mda5 have focused on its antiviral response, further studies investigating the role of Mda5 in sensing bacterial PAMPs or its ability to interact with other bacterial PAMP-sensing PRRs is warranted. Bacterial infection in teleost fish showed overexpression of Mda5, but the studies mentioned in this review did not address the specific immune genes involved. Knowing this that would help explain the mechanisms of protection elicited during bacterial infections. 3. The absence of RIG-1 in Acanthopterygians poses the question whether this group of teleosts has evolved a different gene that performs viral sensing, especially, for short dsRNAs. Studies examining the activity of Mda5 (and LGP2) in this group of teleosts are needed to establish if these two RLRs are able to sense all types of viral RNAs in fish lacking RIG-1.
In summary, the fish innate immune system is not as simple as often described, and although it is less complex than the mammalian immune system, it has evolved many similar defense mechanisms that are present in terrestrial organisms. Future elucidation of the regulatory mechanism of Mda5 during pathogen infection is required for a more comprehensive understanding of the role of this and other PRRs in the immune response of fish.

AUTHOR CONTRIBUTIONS
JL designed and wrote the draft. KT contributed by reading and giving several discussion. TJ organized and finalized the paper.


extfieldsofstudy: Medicine Chemistry

text:
Clobetasol propionate enhances neural stem cell and oligodendrocyte differentiation

Clobetasol propionate (Clo) is a potent topical glucocorticoid and a potential remyelinating agent that has been approved by the U.S. Food and Drug Administration. However, the effect of Clo on neural stem cells (NSCs) remains largely unknown. The aim of the present study was to investigate the effect of Clo on the differentiation of NSCs in vitro. NSCs were isolated from mouse embryonic brain tissues and expanded in vitro. The effect of Clo on NSC viability was examined using an MTT assay. Differentiating NSCs were treated with 5 or 10 µM Clo, or with DMSO control, and the degree of differentiation was examined following culture in stem cell differentiation induction medium for 7 days. The effect of Clo on NSC differentiation was assessed using immunocytochemistry and western blot analyses. The results revealed that Clo significantly increased NSC viability compared with the DMSO control group. Treatment with Clo also significantly increased the number of NSCs that differentiated into growth associated protein 43 positive neurons and corresponding axon lengths were also significantly increased. In addition, treatment with Clo significantly increased the number of myelin basic protein positive oligodendrocytes and decreased the number of glial fibrillary acidic protein positive astrocytes. Furthermore, inhibition of the sonic hedgehog and AMP-activated protein kinase signaling pathways inhibited Clo-induced NSC differentiation, and treatment with Clo upregulated the expression of several neurotrophic factors. In conclusion, the results of the current study suggest that Clo may have a potential therapeutic benefit in neurological disorders affecting oligodendrocytes and neurons.

Introduction
The central nervous system (CNS) does not spontaneously regenerate following injury, a theory that has been widely accepted since it was initially devised in the early 20th century, by the Spanish neuroanatomist Santiago Ramon y Cajal (1). The lack of axonal regeneration in the adult mammalian CNS, particularly in the spinal cord, is due to a combination of factors, including demyelination and injury-induced glial scars (2). Following spinal cord injury, the microenvironment of the spinal cord and brain contains endogenous neural stem cells (NSCs); however, there is no evidence of de novo neurogenesis upon injury (3). Numerous studies have demonstrated that the fate of NSCs is regulated locally by the niche microenvironment and that this is influenced by a number of factors, including specific hormones, such as dexamethasone, growth hormone and prolactin (4,5). Numerous compounds and hormones have been identified to affect NSC differentiation and proliferation (6,7), which suggests that regeneration of the injured spinal cord may be possible.
NSCs are multipotent cells capable of self-renewal and differentiation into multiple cell types, including neurons, astrocytes and oligodendrocytes (8). As such, they are a powerful research tool for examining the mechanisms underlying CNS diseases, as well as therapeutic strategies. Mature NSCs contribute to new memory formation and mediate the response to neural injury (9,10). Therefore, NSC proliferation and differentiation serve an important role in neural development, maintenance and regeneration. However, there is a tendency for NSCs to differentiate into astrocytes, which may form glial scars that suppress spontaneous remyelination and neural recovery (11). Therefore, a compound that promotes endogenous NSC proliferation and differentiation is likely to also promote neuronal repair in the CNS.
Clobetasol propionate (Clo), which has been approved by the U.S. Food and Drug Administration, is a corticosteroid and a member of the glucocorticoid family that is widely used to treat a number of skin disorders, including herpes labialis, psoriasis and lichen sclerosus (12). Clo is also a potential remyelinating agent that has been demonstrated to promote differentiation in oligodendrocyte precursor cell (OPC) cultures and remyelination in vivo (12)(13)(14)(15). Clo enhances the differentiation of ectodermal stem cell-derived OPCs into oligodendrocytes, which has been validated in vivo (16,17).
Clo promotes the proliferation of primary neuronal precursor cells alone and synergistically in the presence of sonic hedgehog (SHH) protein (18). Therefore, Clo may function as an important modulator for the proliferation and differentiation of NSCs; however, to the best of the authors' knowledge, no study to date has examined the direct effect of Clo on cultured NSCs.
The aim of the present study was to determine the effect of Clo on the differentiation of NSCs into neurons, oligodendrocytes and astrocytes in vitro. The results of the current study demonstrate that Clo has potential as a disease-modifying therapy that may have applications in neurological disorders that affect neurons and oligodendrocytes.

Materials and methods
Cell culture. Pregnant (E14) adult Sprague-Dawley (SD) rats (n=5; weight, 250 g; age, 12 weeks; supplied by the Animal Center of Jiangsu University) were used in the present study. The present study, including all animal experimental protocols, was approved by the Jiangsu University Animal Care Committee (Jiangsu, China; protocol number 2016-08-07). Pregnant (E14) SD rats were sacrificed by the intraperitoneal injection of pentobarbital (200 mg/kg). Embryonic rats were dissected from the uterine horn under sterile conditions and placed in PBS. The embryonic brains were immediately harvested by microdissection, and maintained in PBS prior to dissociation. Briefly, cells were dissociated by mechanical shearing followed by filtration through a 40-µm mesh. Cell suspensions were prepared, and primary embryonic neurospheres cultured as previously described (19). The cells were seeded in ultra-low-attachment culture plates (Corning Inc.) and cultured in serum-free DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc) supplemented with 2 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.), 20 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor (both PeproTech, Inc.), 2% B27 (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Beijing Solarbio Science & Technology Co., Ltd.). All cultures were maintained at 37˚C in a humidified atmosphere of 5% CO 2 . After passage 3, NSCs were identified by their morphological characteristics and nestin expression, as described below. The subcultured neurospheres were characterized by immunofluorescence staining with anti-nestin antibody. Neurospheres formed from NSCs of passage three were used for subsequent experiments.
Cell viability assay. The cell viability of NSCs was assessed using the MTT colorimetric assay (Beijing Solarbio Science & Technology Co., Ltd.). Briefly, dissociated neurosphere-derived cells from primary cultures were seeded onto non-treated 96-well plates (Corning Inc.) at a density of 5x10 3 cells/well and grown for 48 h following treatment with different concentrations (2.5, 5, 7.5, 10, 12.5 and 15 µM) of Clo (Selleck Chemicals), dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) to a final concentration of 0.01%, or DMSO alone as a control. After the 48-h treatment, the Clo-containing medium was removed, the cells were gently washed twice with PBS and then 200 µl MTT (0.5 mg/ml) in PBS was added to each well. Following incubation with MTT for 4 h at 37˚C, the formazan dye in DMSO produced by viable cells was quantified by measurement of the absorbance at 460 nm using a microplate reader. Five independent experiments were conducted. In each experiment, 15 replicates were measured for each concentration.

NSC differentiation.
In all experiments NSCs were cultured in serum-free DMEM/F12 for 12 h, unless otherwise stated. For NSC differentiation assays, 100-µm-diameter neurospheres were collected by centrifugation (1,000 x g for 10 min at 4˚C) and 10-15 neurospheres were seeded onto a 48-well plate (BD Biosciences) in stem cell differentiation induction medium [DMEM/F12 containing 4% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 2% B27 and 2% N2]. Following 12-h culture, differentiating NSCs were treated with Clo (5 or 10 µM) or DMSO (control) at 37˚C and differentiation was evaluated after culture in stem cell differentiation induction medium after 7 days of treatment.

SHH inhibition.
To establish whether the SHH signaling pathway is involved in NSC differentiation, the SHH signaling pathway inhibitor cyclopamine (CYC; Sigma-Aldrich; Merck KGaA) was used to treat NSCs. Prior to inhibition experiments, NSCs were cultured in stem cell differentiation induction medium and treated with Clo (10 µM) for 7 days, as described above. Cells were treated with 10 µM CYC diluted in stem cell differentiation induction medium for 7 days.

5'-AMP-activated protein kinase (AMPK) inhibition.
To establish whether the AMPK signaling pathway is involved in NSC differentiation, the AMPK signaling pathway inhibitor Compound C (CC; Sigma-Aldrich; Merck KGaA) was used to treat NSCs. NSCs were cultured in stem cell differentiation induction medium and treated with Clo (10 µM) for 7 days, as described above. Cells were treated with 5 µM CC diluted in stem cell differentiation induction medium for 7 days.

Immunocytochemistr y and western blot analysis.
Immunocytochemistry was used to determine the expression pattern of differentiated cell markers in NSCs. Cells were washed three times with PBS and fixed with 4% paraformaldehyde at 4˚C for 12 h. Subsequently, cells were blocked with For western blot analysis, total protein from adherent cells in each experimental group was harvested. Total protein was extracted from cells using radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing PMSF, phosphatase inhibitor cocktail and EDTA (all Beijing Solarbio Science & Technology Co., Ltd.). Total protein was quantified using a Bradford protein assay and 15 µg protein/lane was separated via SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked for 1 h at room temperature with 3% non-fat dried milk powder in TBS at room temperature. The membranes were incubated with primary antibodies against: GAP-43 (rabbit polyclonal; cat. nos. sc-81492 and sc-514302, respectively) from Santa Cruz Biotechnology, Inc., overnight at 4˚C. Following primary antibody incubation, membranes were incubated with HRP-conjugated secondary antibodies (1:200; cat. nos. BA1050 and BA1056; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature. Protein bands were visualized using an ECL kit (EMD Millipore). Chemiluminescent signals were captured digitally using a chemiluminescence imaging system. The images were analyzed using ImageJ software (version 1.51 k; National Institutes of Health).
Statistical analysis. Data are presented as the mean ± standard error of the mean. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp.,). One-way analysis of variance followed by protected Fisher's least significant difference post hoc test was used to analyze differences among multiple groups. P<0.05 were considered to indicate a statistically significant difference.

Results
Characterization of NSCs. Primary NSCs formed neurospheres of various sizes, with diameters of 50-100 µm after 7 days of culture in serum-free medium (Fig. 1A). To characterize NSCs, immunocytochemical staining of nestin, a commonly used NSC marker, was performed (Fig. 1B). Strong staining of nestin was observed in the cultured neurospheres.

Effect of Clo on NSC proliferation.
To determine the effect of Clo on NSCs, cell viability was examined using the MTT assay following treatment with different concentrations (2.5,    Fig. 2A). However, cell death was observed following treatment with Clo at concentrations ≥12.5 µM. These data indicate that treatment with Clo increases NSC proliferation at low concentrations. Similarly, treatment with Clo significantly increased neurosphere formation at lower concentrations, with a peak number of neurospheres obtained with a Clo concentration of 10 µM (Fig. 2B).

Effect of Clo on NSC differentiation.
To study the effect of Clo on NSC differentiation, NSCs were cultured in specific differentiation induction medium with or without Clo (5 and 10 µM). NSC differentiation was confirmed by immunocytochemistry using neural cell lineage-specific antibodies, which included GAP-43 (neurons), GFAP (astrocytes) and MBP (oligodendrocytes; Fig. 3A). Treatment with Clo significantly increased the number of GAP-43 positive neurons compared with the DMSO control group (Fig. 3B). In addition, treatment with Clo significantly increased the axon length in GAP-43 positive neurons compared with the DMSO control group (Fig. 3C). No significant difference in axon length was observed between the 5 and 10 µM groups (Fig. 3C). No significant difference was detected in astrocyte differentiation between the 5 µM Clo group and the DMSO control group (Fig. 3D). However, treatment with a higher concentration (10 µM) of Clo significantly decreased the number of differentiated GFAP positive astrocytes compared with that in the 5 µM Clo or DMSO control group (Fig. 3D). Treatment with Clo significantly increased the number of MBP positive oligodendrocytes compared with that in the DMSO control group (Fig. 3E). No significant difference was observed between the 5 and 10 µM groups (Fig. 3E).
Furthermore, the protein expression levels of GAP-43, MBP and GFAP were determined by western blot analysis in differentiated NSCs following treatment with Clo (5 or 10 µM) or DMSO (Fig. 4A). Treatment with Clo significantly increased GAP-43 and MBP protein expression levels, and significantly decreased GFAP protein expression levels compared with the corresponding levels in the DMSO control group (Fig. 4B).

Effect of Clo on p-AMPK protein expression.
Given the role of ERK and AMPK signaling pathways in stem cell proliferation and differentiation (20,21), the expression of SHH, p-AMPK and p-ERK proteins was examined in NSCs cultured in specific differentiation induction medium with or without Clo (5 and 10 µM) using immunocytochemistry (Fig. 5A) and western blotting ( Fig. 5B and C). No significant difference in the level of p-ERK was observed between the Clo and DMSO treatment groups (P>0.05; Fig. 5B and C). However, western blot analysis demonstrated that the level of p-AMPK was significantly increased in differentiating NSCs following treatment with Clo compared with the DMSO control group (P<0.05; Fig. 5B and C).

Effect of Clo on SHH and SMO protein expression.
The protein expression levels of SHH and SMO were determined in differentiated NSCs following treatment with Clo (5 or 10 µM) or DMSO (Fig. 5). Immunocytochemistry indicated that treatment with Clo markedly increased SHH protein expression levels (Fig. 5A), and western blotting confirmed that SHH and SMO were significantly increased in the Clo treatment groups compared with the DMSO control group (Fig. 5B and C). These results suggest that treatment with Clo affects SHH signaling and this may provide a mechanistic explanation for the enhanced differentiation of NSCs into neurons and oligodendrocytes in the presence of Clo.
Inhibition assays. To further examine the involvement of AMPK and SHH signaling pathways in NSC differentiation, the SHH pathway inhibitor CYC and the AMPK pathway inhibitor CC were used to block these pathways and NSC differentiation was examined. The protein expression levels of GAP-43, MBP, GFAP, SHH and SMO were determined by western blot analysis in differentiated NSCs following treatment with 10 µM Clo and 10 µM CYC (Fig. 6A). Treatment with Clo alone significantly increased GAP-43, MBP, SHH and SMO protein expression levels, whilst GFAP protein expression levels were significantly decreased compared with those in the DMSO control group (Fig. 6B). However, these Clo-induced changes were attenuated by treatment with CYC,   Immunostaining of SHH, p-AMPK or p-ERK with DAPI nuclear staining in differentiated NSCs following treatment with Clo (5 or 10 µM) or DMSO (control). Scale bar=25 µm. (B) Representative western blots of SMO, SHH, p-ERK, ERK, p-AMPK and AMPK in differentiated NSCs following treatment with Clo (5 or 10 µM) or DMSO (control). (C) Quantification of SMO, SHH, p-ERK/ERK and pAMPK/AMPK protein levels. Data are presented as the mean ± standard error of the mean. n=5. * P<0.05 and ** P<0.01 vs. DMSO. Clo, clobetasol propionate; SMO, smoothened homolog; SHH, sonic hedgehog; AMPK, AMP-activated protein kinase; ERK, mitogen-activated protein kinase 1; p, phosphorylated; NSCs, neural stem cells. with no significant difference from the DMSO control group being detected (Fig. 6B).
Furthermore, the protein expression levels of GAP-43, MBP, GFAP, AMPK and p-AMPK were determined by western blot analysis in differentiated NSCs following treatment with 10 µM Clo and 5 µM CC (Fig. 7A). Treatment with Clo alone significantly increased GAP-43, MBP and p-AMPK protein expression levels, whilst GFAP protein expression levels were significantly decreased compared with those in the DMSO control group (Fig. 7B). However, these Clo-induced changes were attenuated by treatment with CC, with no significant differences detected compared with the DMSO control group (Fig. 7B).
Effect of Clo on brain-derived neurotrophic proteins. The protein expression levels of the neurotrophic factors NT-3 and BDNF were determined by western blot analysis in differentiated NSCs following treatment with Clo (5 or 10 µM) or DMSO (Fig. 8A). Treatment with Clo significantly increased NT-3 and BDNF protein expression levels compared with those in the DMSO control group (Fig. 8B).

Discussion
The present study demonstrated that Clo significantly enhanced NSC viability, and promotes the differentiation of NSCs into neurons and oligodendrocytes whilst inhibiting astrocyte differentiation. Furthermore, the present study indicated that Clo may promote NSC differentiation through the SHH and AMPK signaling pathways, providing a potentially novel mechanism underlying the therapeutic effect of Clo in CNS injury.
Previous studies demonstrated that Clo enhances differentiation in OPC cultures (17) and reduces myelin loss (22). However, whether Clo also affects NSC differentiation was unknown. Therefore, the aim of the present study was to investigate the effect of Clo on the differentiation of NSCs in vitro. NSCs were isolated from mouse embryonic brain tissue and expanded in vitro. It was observed that the NSCs, cultured as neurospheres, expressed nestin, a widely used marker of NSCs (23). Neurosphere formation can reflect the self-renewal capacity of cells (4). The MTT assay in the present study revealed that treatment with Clo significantly increased  NSC viability at low concentrations (2.5, 5, 7.5 and 10 µM), consistent with a previous study (18). However, cell death was observed following treatment with Clo at concentrations ≥12.5 µM, indicating that high concentrations Clo may be toxic to NSCs. In addition, a recent study revealed that Clo activates the SHH signaling pathway, and as high concentrations of SHH inhibit cellular proliferation (24), high concentrations of Clo may inhibit NSC viability in a similar manner.
A previous study identified four glucocorticoids (halcinonide, fluticasone propionate, Clo and fluocinonide) as SMO agonists via a high-throughput screen of cells containing an SMO and β-arrestin-GFP reporter that binds to activated SMO (18). SMO is a component of the SHH signaling pathway (25), which can promote the expression of HH target genes by the Gli family of transcription factors (26). SHH is a morphogen produced by the notochord and the ventral floor plate that induces the development of various populations of ventral neurons and oligodendrocytes, and the proliferation of embryonic spinal cord OPCs and NSCs (27). SHH also serves a role in the development and maintenance of the nervous system (28). The current study demonstrated that treatment with Clo upregulates the expression of SMO and SHH proteins in differentiating NSCs. Although the molecular mechanism of Clo remains unknown, it can be hypothesized that Clo may activate the SHH signaling pathway by targeting SMO. Najm et al (17) reported that the glucocorti-17) reported that the glucocorti-) reported that the glucocorticoid receptor may be involved in the process that regulates NSC differentiation into oligodendrocytes; however, the underlying molecular mechanism requires further investigation.
A previous study has demonstrated that Clo acts as a SMO agonist to promote myelin gene expression (18). Therefore, the Clo-associated enhancement of NSC viability and differentiation observed in the present study may be due to Clo having SHH-like and SMO agonist-like effects. Given that Clo is able to pass through the blood-brain barrier (16), Clo may exert a direct effect on the CNS. To further investigate whether Clo promotes the differentiation of NSCs through SHH signaling, SHH signaling was blocked in differentiating NSCs using CYC (29). Blocking the SHH signaling pathway exhibited a marked effect on Clo-induced NSC differentiation. Inhibition experiments further confirmed that Clo enhances NSC differentiation through the SHH signaling pathway.
Furthermore, the present study indicated that the AMPK signaling pathway may be involved in NSC proliferation and differentiation, as it was significantly upregulated following treatment with Clo. It is thought that AMPK maintains cellular energy homeostasis through the regulation of glucose and lipid metabolism (30). Although AMPK activation may inhibit cell proliferation (31,32), the current study revealed that treatment with Clo enhances NSC viability. AMPK was previously demonstrated to be expressed in nerve cells (33). In the current study, p-AMPK was detected at elevated levels in differentiated NSCs. Inhibition experiments confirmed that the involvement of the AMPK signaling pathway in the regulation of NSC differentiation. A previous study reported that the administration of glucocorticoids may cause hyperphagia via the AMPK-neuropeptide Y (NPY) signaling pathway (34). NPY is involved in the modulation of the dynamics of NSC niches in the dentate gyrus and subventricular zone. Therefore, it may be hypothesized that AMPK-NPY is a target for Clo in NSCs. SHH signaling promotes polyamine biosynthesis in cerebellar granule cell precursors and this process is governed by AMPK (35). Although the mechanism underlying the activation of AMPK signaling by Clo was not elucidated in the current study, it is possible that SHH may be involved. Furthermore, it was previously revealed that Clo is able to upregulate the neural cell expression of NT-3 and BDNF, to promote neural differentiation and proliferation (36). Hence, Clo may enhance NSC differentiation and proliferation via the upregulation of neurotrophic factors.
The present study demonstrated that a high concentration (10 µM) of Clo significantly suppressed the differentiation of NSCs into astrocytes. This was likely caused by the ability of corticosteroids to inhibit the inflammatory response in cells (37). Neuroinflammation is a feature associated with CNS disorders, including spinal cord injury and neurodegeneration, in which astrocytes serve critical roles (38). The reduced astrocyte differentiation capacity of Clo-treated NSCs may be a potential benefit towards the reduction of astrogliosis, the main cause of glial-scar formation, which limits axonal outgrowth and regeneration.
The current study has several limitations. In order to validate the results obtained in the current study, relevant in vivo studies and further mechanistic studies are required. In addition, human-derived stem cells should be used to ensure there are no species-specific differences.
In conclusion, the current study identified a novel, straightforward and efficient method to obtain differentiated cells with the typical morphological characteristics associated with NSC-derived neurons and oligodendrocytes. Furthermore, the well-known pharmacokinetic and pharmacodynamic properties of Clo combined with the results of the present study support the potential therapeutic application of Clo in human regenerative medicine. reviewed and edited the manuscript. All authors read and approved the fi nal manuscript.

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s2fieldsofstudy: Biology Medicine

text:
Dedicator of Cytokinesis 4: A Potential Prognostic and Predictive Biomarker Within the Metastatic Spread of Breast Cancer to Bone

Metastasis to bone occurs in over 70% of patients with advanced breast cancer resulting in skeletal complications, including pathological fractures, hypercalcaemia, and bone pain. Significant advances have been made in the treatment of bone metastases, including the use of antiresorptive drugs, such as bisphosphonates, as well as antibody-based therapies targeting key signalling intermediates within the process of cancer-mediated bone destruction. Despite these advances, treatment is not without side effects, including osteonecrosis of the jaw therefore biomarkers predictive of which patients are at high risk of developing bone spread are required to enable personalized medicine initiatives within this important disease area. We used proteomic analysis to compare the protein expression within (1) a parental triple negative human breast cancer cell line, (2) a fully bone homing cell line and (3) a lung homing cell line. The bone and lung homing cell-lines were derived by intra-cardiac injection of fluorescently labelled cells within immune-compromised mice. Proteomics identified Dedicator of Cytokinesis 4 as a biomarker predictive of bone spread, and this finding was further supported by the observation that high levels of Dedicator of Cytokinesis 4 within primary breast tumours were predictive of breast cancer spread to bone. Here, we provide an overview of this study and put the findings into context.

In our recent publication, 1 we have described the discovery of the role of Dedicator of Cytokinesis 4 (DOCK4) as a potential biomarker protein predictive of bone metastasis within breast cancer. Bone is a common site of metastasis within advanced breast cancer, and over 70% of advanced breast cancer patients develop bone metastases. 2 Patients present with symptoms of bone metastasis in many cases years after apparently successful treatment, with disseminated tumour cells remaining dormant within bone prior to their activation. The cellular mechanisms leading to both breast cancer metastasis to bone, and the eventual reactivation of dormant breast cancer cells, are currently the subject of intensive research. Bone is the subject of constant turnover with bone homeostasis being regulated by the activity of bone-resorbing osteoclasts and bone-forming osteoblasts. 3 Disseminated and reactivated breast cancer cells secrete parathyroid hormone-related protein (PTHrP), which triggers osteoblasts to increase their synthesis and secretion of receptor activator of NF-κB ligand (RANKL), as well as reduce their secretion of osteoprotegerin (OPG) (an orphan receptor for RANKL). This results in increased binding of RANKL to the receptor for RANKL (RANK) resulting in osteoclast activation and bone destruction. Degradation of bone releases growth factors trapped in the bone matrix, which in turn stimulate breast cancer cell proliferation and release of PTHrP, resulting in a 'vicious cycle' of bone destruction. 4 Current treatments for bone metastasis within breast cancer include bisphosphonate drugs (such as zoledronic acid) 5 and antibody-based therapies, eg, denosumab. 6 These treatments have greatly improved patient quality of life, with zoledronic acid conferring a statistically significant improvement in invasive disease-free survival within women who were >5 years post-menopausal. 7 Despite these improvements in patient treatment, these drugs are not without side effects, including the case of bisphosphonate treatment of osteonecrosis of the jaw. 8 To date, there are no effective biomarkers available to predict which patients are at greatest risk of developing bone spread, enabling a personalised medicine approach towards the treatment of skeletal metastasis within breast cancer.
We are engaged in research aiming to elucidate the molecular basis of cancer spread to the skeleton. This work has benefitted from access to unique patient sample banks, including samples from the large, open-label, international, multicentre, randomised, controlled, parallel-group phase 3 trial AZURE, which asks the question 'Does adjuvant zoledronic acid reduce recurrence in patients with high risk localised breast cancer?'. The AZURE trial has provided both tissue samples (tissue microarrays) as well as patient serum and plasma samples from patients receiving standard adjuvant therapy either with or without addition of zoledronic acid. In addition, proteomic methods have been used to characterise murine models of breast cancer spread to bone, including intracardiac injection models of bone metastasis. 9 Our proteomic analysis of bonehoming and parental triple negative breast cancer cells derived from intracardiac injection murine models has identified a 2 Cancer Informatics panel of potential bone metastasis biomarker candidates including macrophage actin-capping protein (CapG), the PDZ-domain containing adaptor protein GIPC1, 10 and the cell motility regulator DOCK4. 1 DOCK4 was identified as a potential biomarker predicting bone metastasis risk via quantitative proteomic analysis of bone-homing and parental MDA-MB-231 cells using stable isotope labelling by amino acids in cell culture (SILAC). 1,11,12 DOCK4 expression was elevated 2.5-fold within bone homing cells and was not elevated within a lung-homing control cell line, suggesting bone specificity. Immunohistochemical assessment of DOCK4 expression levels within AZURE-patient derived tissue microarrays (n = 689) demonstrated that DOCK4 levels were significantly prognostic for first recurrence within bone in control arm standard chemotherapy) patients (hazard ratio [HR]: 2.13, 95% confidence interval [CI[: 1.06-4.30, P = .034), an effect not observed within the zoledronate treatment arm (HR: 0.812, 95% CI: 0.176-3.76, P = .790). 1 There was no association of DOCK4 levels with metastatic spread to nonbone sites suggesting that DOCK4 levels within primary breast tumours predict aggressive disease and risk of metastasis specifically to bone. The absence of a predictive effect within the zoledronate-treated arm suggests that DOCK4 levels may also inform patient treatment decisions. 1 There was no statistically significant difference in overall survival between patients with high and low DOCK4 levels within this study.
Our study aimed primarily to look into the role of DOCK4 as a potential predictive marker for bone metastasis. Within our study, high DOCK4 levels reduced the incidence of nonskeletal metastases within patients in the control arm of the AZURE trial. 1 This effect was not observed within the zoledronic acid treatment arm, suggesting that zoledronate may remove an advantageous effect of high DOCK4 levels upon the incidence of nonskeletal metastases. The mechanism of action of zoledronate on nonbone metastases is currently unknown; however, it has been previously described that zoledronic acid can inhibit the proliferation, survival, and migration of breast cancer cells. 13 DOCK4 levels may therefore have prognostic potential for nonskeletal as well as skeletal metastases.
The role of DOCK4 as a prognostic biomarker for metastatic spread to bone was a unique finding within our published study. DOCK4 is a known guanine nucleotide exchange factor (GEF) responsible for activation of several GTPases inside the cell, including the GTPase protein Rac1, a pivotal regulator of cell migration expressed at the leading edge of motile cells. 14 The mechanism of action of DOCK4 within the regulation of cell migration is still the subject of active research. Previous studies have demonstrated that the proangiogenic growth factor vascular endothelial growth factor (VEGF) promotes the interaction of DOCK4 with DOCK9 (a GEF for the GTPase Cdc42). 15 In addition, VEGF-dependent Rac activation by DOCK4 is required for the activation of the GTPase Cdc42, and VEGF-dependent Rac activation is itself dependent on the activation of a further GTPase RhoG. 15 In this way, the activity of a GTPase signalling cascade functions to both activate DOCK4 and transduce signals downstream of this important GEF. DOCK4 activation leads to the formation of lateral filopodia within epithelial cells within tumours, and the formation of blood vessel lumen within tumour angiogenesis. 15 DOCK4 thus plays a pivotal role within tumour angiogenesis; however, its precise role within bone-specific metastasis is still to be elucidated.
While elucidating the role of DOCK4 within cancer spread to bone, we must be aware that proteins often play pleiotropic roles within the cell. Research within our group previously identified actin capping protein (CapG) as a prognostic biomarker for risk of breast cancer spread to bone. Actin-capping proteins do not appear to offer an obvious bone-specific function for metastatic spread; however, subsequent research revealed that CapG can act as an epigenetic regulator responsible for the increased expression of a prometastatic gene stanniocalcin-I (STC-I), required for metastasis to bone. 16 Discovered biomarker proteins may thus achieve their effects via functions that differ from the primary gene annotation, and, thus, the pro-bone-metastatic role of DOCK4 may involve additional functions to the regulation of cell motility via GTPases.
The expression of DOCK4 is induced by the cytokine transforming growth factor-β (TGFβ), a pivotal cytokine within bone metastasis. 17 TGFβ acts via Smad phosphorylation to increase the expression of numerous gene products, including c-MAF, a transcription factor responsible for promoting bone metastasis within breast cancer. 18 Previous research within our group has also identified GIPC1 (a scaffolding protein and binding partner of the TGFβ receptor) as a potential biomarker prognostic for increased risk of bone metastasis within breast cancer. 10 DOCK4 expression levels may thus be positively correlated with the expression levels of c-MAF, 19 suggesting that, within bone metastasis of breast cancer, TGFβ increases expression of both c-MAF and DOCK4. DOCK4 may thus act as a functional mediator of a c-MAF response panel. The precise role of DOCK4 function within metastasis to bone is still to be elucidated. Studies conducted within our laboratory demonstrated that stable knockdown of DOCK4 expression reduced breast cancer cell motility; however, this effect was transient, being significant at 6 hours post knockdown, but not observed as significant levels 12 hours post knockdown. 1 Cell motility may be the only aspect of DOCK4's role that is altered in bone metastasis of breast cancer.
Our research to date has clearly identified a series of proteins with elevated expression specifically within bone metastasis of breast cancer. Functional study of these proteins has Brown et al 3 identified that they are frequently downstream of key cytokines and growth factors within skeletal metastasis, including TGFβ. We anticipate that DOCK4 will be just one protein within a panel of proteins ultimately forming a prognostic test for clinical application. Multiprotein tests have greater utility for informing patient treatment than single-protein tests, 20 and have a greater ability to deal with diseases with known molecular heterogeneity, such as breast cancer. Research efforts are therefore currently underway to further supplement our protein panel with additional proteins, thus increasing the sensitivity and specificity of this clinical test when eventually applied. There are many aspects of DOCK4's role within bone metastasis of breast cancer, which remain to be elucidated, including the possible role of DOCK4 within nonbone metastasis, as well as the interaction of DOCK4 with bisphosphonate treatment and its potential role as an informative biomarker for patient treatment decisions.

Author Contributions
SLW and JEB drafted and reviewed the manuscript. SLW, JAW and JEB were involved in experimental design and data analysis.