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Muscle Expression of SOD1G93A Modulates microRNA and mRNA Transcription Pattern Associated with the Myelination Process in the Spinal Cord of Transgenic Mice

A crucial system severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. One of the best examples of impaired interplay between muscle and nerve is Amyotrophic Lateral Sclerosis, a neurodegenerative disease characterized by degeneration of motor neurons and muscle atrophy. Increasing evidences suggest that damage to motor neurons is enhanced by alterations in the neighboring non-neuronal cells and indicate that altered skeletal muscle might be the source of signals that impinge motor neuron activity and survival. Here we investigated whether muscle selective expression of SOD1G93A mutant gene modulates mRNAs and miRNAs expression at the level of spinal cord of MLC/SOD1G93A mice. Using a Taqman array, the Affymetrix Mouse Gene 2.0 ST approach and the MiRwalk 2.0 database, which provides information on miRNA and their predicted target genes, we revealed that muscle specific expression of SOD1G93A modulates relevant molecules of the genetic and epigenetic circuitry of myelin homeostasis in spinal cord of transgenic mice. Our study provides insights into the pathophysiological interplay between muscle and nerve and supports the hypothesis that muscle is a source of signals that can either positively or negatively affect the nervous system.

INTRODUCTION
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder in which the functional connection between nerve and muscle is severely compromised (Musarò, 2013). ALS affects pyramidal neurons in the motor cortex and lower motor neurons originating in the brainstem and in the spinal cord. The most typical feature of this progressive lethal disease is the degeneration of motor neurons, muscle weakness and atrophy, that leads to progressive paralysis and death by respiratory failure in few years (Musarò, 2010(Musarò, , 2013. ALS is epidemiologically classified into two major forms: sporadic (90-95%) and familial (5-10%) form. Among the familial cases, ∼20% are caused by dominantly inherited mutations in the Cu/Zn superoxide dismutase (SOD1) protein (Rosen, 1993) a cell scavenger of superoxide.
One of the critical issue that remains to be addressed in ALS is whether motor neurons are the first and sole direct targets of ALS or if the toxic activity of mutant SOD1 is cell autonomous. Recent experimental evidence revealed that ALS is a multi-factorial and multi-systemic disease in which alterations in structural, physiological, and metabolic parameters in different cell types, such as motor neurons, glia, and muscle cells, may act synergistically to exacerbate the disease (Boillée et al., 2006;Julien, 2007;Musarò, 2013). Skeletal muscle is a source of signals that influence neuron survival, axonal growth and maintenance of synaptic connections. Notably, development in the absence of skeletal muscle results in the sequential ablation of motor neurons from the spinal cord to the brain (Kablar and Rudnicki, 1999); thus, effective connection between muscle and nerve is crucial for the survival and function of both tissues throughout life. We have previously demonstrated that muscle-specific expression of SOD1 mutation in the MLC/SOD1 G93A transgenic mouse model, which selectively expresses the mutant SOD1 G93A gene in the skeletal muscle under the transcriptional control of a muscle-specific promoter (MLC), induces accumulation of Reactive Oxygen Species, causes muscle atrophy with a concomitant alteration in the ultrastructure and in the functional performance of skeletal muscles, and promotes microglia activation in the spinal cord, that is a presymptomatic sign of ALS disease (Dobrowolny et al., 2008b). Moreover, it has been reported that skeletal muscle-restricted expression of the human mutant SOD1 gene causes motor neuron degeneration in old transgenic mice (Wong and Martin, 2010) and that muscle-selective alterations in mitochondrial function initiate neuromuscular junction alteration, distal axonopathy, astrocytosis, and mild motor neuron loss (Dupuis et al., 2009). We also demonstrated that forced expression of muscle specific isoform of the Insulin Growth Factor-1 (mIGF-1) exclusively in the skeletal muscle of the global SOD1 G93A mice counteracted the symptoms of ALS, induced satellite cell activation, stabilized neuromuscular junctions, preserved motor peripheral nerve, and led to a reduction in astrocytosis in the SOD1 G93A spinal cord (Dobrowolny et al., 2005(Dobrowolny et al., , 2008a. All together these data indicate that nervous development and homeostasis are intimately coupled to skeletal myogenesis and muscle function. Moreover, these findings support the hypothesis that skeletal muscle is a primary target of mutant SOD1 toxicity in mice and indicate that altered skeletal muscle impairs motor neuron activity, suggesting a sequential pattern of degeneration in which muscle abnormalities precede motor neuron death. In order to add new insights into the pathogenesis of ALS and define whether skeletal muscle alteration potentially affect the nervous system we analyzed the mRNA and miRNA expression in the lumbar ventral spinal cord of MLC/SOD1 G93A transgenic mouse model (Dobrowolny et al., 2008b). Interestingly, we found that muscle specific expression of SOD1 G93A gene affects spinal cord miRNome and transcriptome; in particular we observed the activation of genes involved in the myelination process and a decrease in the axon diameter to total fiber diameter in the sciatic nerve, suggesting a myelinopathy in the transgenic mouse model.

Mice
Animal model used: 4 month old MLC/SOD1 G93A mice overexpressing the mutant SOD1 gene (SOD1 G93A ) under the control of the Myosin Light Chain (MLC) muscle specific promoter (Dobrowolny et al., 2008b) and 4 month old FVB (Friend leukemia virus B) (control strain). The animals were housed in a temperature-controlled (22 • C) room with a 12:12 h light-dark cycle. All animal experiments were approved by the ethics committee of the Unit of Histology and Medical Embryology-Sapienza University of Rome-and were performed in accordance with the current version of the Italian Law on the Protection of Animals.

RNA Isolation
Total RNA was isolated from frozen lumbar spinal ventral tissue specimens using pestel homogenization, TRIzol reaction (Invitrogen, Carlsbad, CA, USA), and further on-column purification as previously described. The yield, quality and integrity of RNA were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as previously described (Bernardini et al., 2012).

miRNA Analysis
From each sample (n = 4 per experimental group), 600 ng of total RNA was reverse transcribed using Taqman MicroRNA Reverse Transcription Kit with Megaplex Primer Pools A & B (Applied Biosystems, Foster City, CA, USA). The cDNA was analyzed with Taqman Array Mouse MicroRNA A and B Cards Set version 3 (Applied Biosystems, Foster City, CA, USA) on the Applied Biosystems 7900HT qPCR system (Applied Biosystems, Foster City, CA, USA). Most of the MicroRNAs in the B Card were undetermined, therefore their analysis was omitted. The data was normalized by global mean normalization. SDS RQ Manager version 2.4 (Applied Biosystems, Foster City, CA, USA) was used to calculate Ct values (number of cycles required for the fluorescent signal to cross the set threshold), Ct values greater than 35 were considered as non-specific or undetected, and were filtered. MicroRNAs with missing values in at least 2 of 4 replicate samples per treatment group were removed from the dataset.
The remaining Ct values were normalized using the U6 reference miRNA as suggested by manufacturer's protocol. The fold change between sample groups was calculated by subtracting the relative expression of the samples and then transforming the result to anti-log2 (i.e., 2 difference ).

miRNA Pathway Analysis
The putative correlation between mRNAs and miRNAs was investigated through the MiRwalk 2.0, a comprehensive database that provides information on miRNA from Human, Mouse and Rat on their predicted as well as validated binding sites on their target genes. In particular we get information on miRNA produced by 8 established miRNA prediction programs on 3 ′ UTRs of all known genes of Mouse (Dweep et al., 2011;Bernardini et al., 2012). miRNA predicted pathways were identified using DIANA (DNA Intelligence Analysis) tools (Vlachos et al., 2012).
DIANA-miRPath is a miRNA pathway analysis web-server that utilizes predicted miRNA targets (in CDS or 3 ′ -UTR regions) provided by the DIANA-microT-CDS algorithm or even experimentally validated miRNA interactions derived from DIANA-TarBase v6.0. These interactions (predicted and/or validated) can be subsequently combined with sophisticated merging and meta-analysis algorithms. Essentially, this tool allows identifying all the predicted or experimentally validated miRNAs significantly targeting a selected pathway.

Single qPCR Validation
Single qPCR of miRNA 133a, 133b, 9, 29, 330, and 1 was performed to validate the performance of the Taqman arrays using Taqman probes from single tube Taqman microRNA assays with the diluted cDNA in a 10 µl reaction volume, in triplicate for each assay, on the Applied Biosystems 7500HT system (Applied Biosystems, Foster City, CA, USA; n = 10 per experimental group).
Five independent samples for each group of animals have been used for densitometric analysis and protein level of α-tubulin was used as control for equal protein loading.

Microarray Analysis
Total RNA was used to create the biotin-labeled library to be hybridized on GeneChip R Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) covering more than 26500 RefSeq coding transcripts and more than 3500 RefSeq noncoding transcripts, following the manufacturer protocol.
Briefly, double-stranded cDNA was synthesized routinely from less than 1 microgram of total RNA primed with a poly-(dT)-T7 oligonucleotide. The cDNA was used in an in vitro transcription reaction in the presence of T7 RNA polymerase and biotin-labeled modified nucleotides during 16 h at 37 • C. Biotinylated cRNA was purified and then fragmented (35-200 nucleotides), together with hybridization controls and hybridized to the microarrays for 16 h at 45 • C. Using the GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA), the biotin-labeled cRNA was revealed by successive reactions with streptavidin R-phycoerythrin conjugate, biotinylated anti-streptavidin antibody and streptavidin R-phycoerythrin conjugate. The arrays were finally scanned in an Affymetrix GeneChip Scanner 7G Plus (Affymetrix, Santa Clara, CA, USA).

Gene Expression Data Analysis-Differentially Expressed Gene List
The CEL files that store the results of the intensity calculations on the pixel values collected from an Affymetrix scanner and result from the hybridization, were analyzed using Transcriptome Analysis Console (TAC) from Affymetrix (Affymetrix, Santa Clara, CA, USA). Gene-level calculation was performed by Robust Multichip Average to summarize probeset signal  and normalization by quantile sketch . A data table (rma), together with the relative CEL and relevant information about the experiment, is available at http:// www.ncbi.nlm.nih.gov/geo/ under accession GSE69582.
An unpaired One-way ANOVA was then used to identify differentially expressed genes, only the genes which met our criterion (p < 0.05, fold change > 1.5) were selected in this study. The resulting gene list was then annotated according to the Gene Ontology (GO) database (www.geneontology.org). This allowed assigning a category to each gene in the list, according to three defined "ontologies" (i.e., terms representing gene product properties): cellular component, biological process and molecular function.
The expression of selected genes was quantified in real time PCR to obtain an independent validation of microarray data. Real time PCR was carried out as previously described elsewhere (Bernardini et al., 2012).

Functional Categorization
The gene expression list has been functionally categorized using the Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/) (Sherman et al., 2007). The algorithms implemented in this software allow identifying over-represented gene ontology (GO) terms with respect to the total number of genes assayed and annotated. To this aim, DAVID applies a modified Fisher exact test, to establish if the proportion of genes falling into an annotation category significantly differs from the background group of genes. In addition, this tool enables the fine mapping of genes within welldefined signaling and/or metabolic pathways, classified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/kegg/). The KEGG mapping tool was employed for the functional categorization of the gene regulatory networks. For this purpose, AffyGene IDs, corresponding to the genes in the selected list, were used as queries and the whole set of genes represented on the array was used as the background group. A p < 0.05 was set.
The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) online software (Franceschini et al., 2013) was used to search interaction relationships of the proteins encoded by differential expressed genes.

Histological Analysis
Sciatic nerves were dissected and quickly fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, washed in 0.1 M sodium cacodylate. Nerves were then post-fixed in 1% osmium in 0.1 M sodium cacodylate for 1 h, dehydrated in ethanol and embedded in epoxy resin. Samples were infiltrated with toluene and cut with a Leica ultracut R ultramicrotome (Leica Microsystems Inc. Buffalo Grove, IL) to obtain 2 um suitable for toluidine blue staining. Microscope using 40 × lenses was employed, and images were processed using Axiovision Rel 4.8 (Zeiss, Oberkochen, D) representative images of sciatic nerve thin sections are shown cropped in Figure 5.
The g-ratio was calculated as (dmax + dmin)/2/(Dmax + Dmin)/2 and the analysis was performed for 200 fibers. Each diameter was calculated from the average of the major axis (Dmax: maximum fiber diameter; dmax:maximum axonal diameter) and the minor axis (Dmin: minimum fiber diameter; dmin: minimum axonal diameter), (Tsutsumi et al., 2014) and was measured using Image J software (Image J 1.36, National Institutes of Health, MD, USA).

Statistical Analysis
Statistical analysis, if not differently specified was performed using the Mann Whitney nonparametric test and significance was established for p < 0.05.

RESULTS
miRNA and mRNA Expression Profile of the Spinal Cord of the MLC/SOD1 G93A Mice First, to identify deregulated miRNAs and transcripts in the spinal cord of MLC/SOD1 G93A transgenic mice we performed miRNA expression profiling and DNA microarray, using the Taqman array card and Affymetrix Mouse Gene 2.0 ST respectively (GSE71194). Among the analyzed miRNAs, 54 were significantly down-regulated (p < 0.05) in the spinal cord of MLC/SOD1 G93A transgenic mice compared to wild type littermates ( Table 1). Of note, the miRnome profiling revealed the down regulation of mir-330, mir-133, and mir-1, which are involved in denervation and reinnervation processes (Jeng et al., 2009;Tsutsumi et al., 2014). In addition mir-29 and mir-9 family member were downregulated and their deregulation was associated with different neurodegenerative diseases, including Huntington, Alzheimer, and Parkinson diseases (Saito and Saito, 2012;Tsutsumi et al., 2014) and demyelination-related diseases (Li and Yao, 2012;Tsutsumi et al., 2014). Real time PCR analysis was performed to validate the expression of these relevant miRNAs in the spinal cord of both wild type and MLC/SOD1 G93A transgenic mice (Figure 1).
The microarray data, related to the expression of mRNA in the spinal cord of both wild type and MLC/SOD1 G93A transgenic mice, were deposited in the GEO database (GSE69582) and the list of the selected significantly up-regulated and downregulated genes (p < 0.05; FC > 1.5) is shown in Tables 2, 3 respectively. Among these genes, nine up-regulated transcripts are involved in the myelination process such as Peripheral myelin protein 22 (Pmp22), Myelin protein zero (Mpz), Periaxin (Prx), the Early growth response 2 (Egr2) genes, Desert hedgehog (Dhh), or encode for extracellular matrix molecules such as Matrilin 2 (Matn2), Gliomedin (Gldn), Claudin 19 (Cldn19), and Smoothelin (Smtn). Other relevant modulated genes include glycoproteins, such as Osteoglycin (Ogn) and dystroglicans, such as Dystrophin related protein 2 (Drp2), associated with the myelination process. The Mpz, Pmp22, Egr2, Prx mRNAs were selected for validation by qRT-PCR analysis and results are shown in Figure 2. Opalin, another gene associated to myelin structures, was down-regulated in the spinal cord of MLC/SOD1 G93A transgenic mice, compared to wild type littermates ( Table 3). Opalin is expressed specifically in late stage of oligodendrocyte differentiation and has been shown to be dramatically reduced in a hypomyelination mouse model (Jiang et al., 2013;Tsutsumi et al., 2014).
In order to show changes in protein expression, as a result of altered microRNA/mRNA expression, we performed western blot analysis for Pmp22 and Mpz expression. We revealed a significant up-regulation of Pmp22 and Mpz proteins in the spinal cord of MLC/SOD1 G93A compared to wild type littermates (Figure 3), further supporting the evidence that these factors are targets of specific microRNAs that control the denervation and reinnervation processes.
Taken together these results suggest that the muscle specific expression of the mutated isoform of the human SOD1 gene alters the expression of relevant markers of myelination in

Functional Annotation of the Identified Genes
Functional annotation of differentially expressed genes was performed using the DAVID functional annotation tool, an integrated biological knowledge base and analytic tools aimed at systematically extracting biological meaning from large gene and protein lists (Huang da et al., 2009). Similar annotation contents were clustered into annotation group with biological meanings. The annotation results revealed that 2.6% of the gene list examined was consistent with OMIM disease categories, in particular 5 of 17 genes extracted were related to Charcot-Marie-Tooth disease. On the other hand, the gene ontology analysis revealed that 39.3% of all gene list are significantly involved in biological processes category, 36.7% are part of cell components, and 39.8% are associated with molecular function categories. The nine interesting genes mentioned above were spread out in all the categories examined. The functional annotation clustering was done using the default parameters, Mus musculus as background and classification stringency was set as medium. Table 4 shows that the significantly modulated genes were involved in 5 functional clusters containing functional annotation for extracellular region, neuron development and differentiation, axonogenesis, and neurological system process. A further functional analysis was performed using STRING, a web base tool to explore potential protein-protein interaction, confirming the interactions among Pmp22, Mpz, Prx, and Egr2 proteins (Figure 4).

Correlation of Expression Profile between miRNAs and mRNA
miRNAs are key regulators of all important biological processes and the level at which they act on coding mRNA (transcriptional, translational, etc.) is still debated; however gene repression by microRNAs at the level of mRNA translation is the most frequently reported mechanism (Morozova et al., 2012). Therefore, we further performed a systematic analysis on the interaction between significantly modulated miRNAs and mRNAs considering only miRNAs present in our list with an inverse mRNAs expression correlation. The analysis was performed using MiRwalk tool a comprehensive database that provides information on miRNA produced by 8 established miRNA prediction programs on 3 ′ UTRs of all known genes of mouse such as RNA22, miRanda, miRDB, TargetScan. Secondly, we ordered mRNAs on the basis of the number of putative targeting miRNAs and we observed that 36 genes were targeted from at least 1 to maximum 30 significantly modulated miRNAs; conversely 54 listed miRNAs targeted at least one to maximum of 11 significantly modulated genes; interestingly the most intriguing cross interaction involved genes and miRNAs of the myelination process (Table 5). This finding clearly suggests that muscle specific overexpression of the SOD1 mutant gene induces the modulation of miRNAs and putative targeted-mRNAs involved in the

Mapping miRNAs to Signaling Pathways
DIANA-mirPath is a web-based computational tool to identify KEGG signaling pathways regulated by miRNAs (see Materials and Methods section). The software compares each set of miRNA targets with all-known KEGG pathway and interestingly we observed high significance for axon guidance, glutamatergic synapse, and amyotrophic lateral sclerosis (ALS), meaning that these pathways are likely to be controlled by the altered miRNAs ( Table 6).

Muscle Specific Expression of SOD1 Mutant Gene Induces Hypomyelination in the Mice Sciatic Nerve
Different reports indicate that ALS might be associated with motor nerve fiber (Echaniz-Laguna et al., 2006;Rajabally and Jacob, 2008;Ahdab et al., 2013). Previously, we demonstrated that the peripheral nerve was severely compromised in the global SOD1 G93A mutant mice, which displayed loss of Schmidt-Lantermann incisures, a disproportionately thick myelin sheath, abundant double onion bulb structures, and increased demyelinated axons (Dobrowolny et al., 2008a). To assess whether muscle specific expression of SOD1 mutant gene induces a peripheral alteration of axon myelination we calculated the ratio of axon diameter to total fiber diameter (g-ratio) (Michailov et al., 2004). The Figure 5A shows the cross-sections of wild type and MLC/SOD1 G93A sciatic nerves stained with toluidine blue. The histological analysis of the peripheral nerve of the MLC/SOD1 G93A displayed the presence of double onion bulb structures. Moreover, the g-ratio in the transgenic sciatic Annotation cluster 7 Enrichment score: 0.53 GOTERM_BP_FAT neurological system process 11 3.9E-2 Annotation cluster means a group of terms having similar biological functions. Enrichment score is the rank of the annotation cluster biological significance. Count column refers to number of genes involved in the same category.
nerves was significantly increased ( Figure 5B) independently of the axonal diameter ( Figure 5C), indicating that muscle specific expression of SOD1 mutant gene induces hypomyelination in the sciatic nerve of transgenic mice.

DISCUSSION
The functional interplay between muscle and nerve is crucial for the survival and function of both tissues throughout life and ALS represents one of the best examples of impaired interplay between the two tissues (Musarò, 2013). Although, there have been significant advances in our understanding of the biology of ALS, no consensus has emerged as to which cells, tissues and pathways are directly affected by mutant SOD1. Motor neurons degeneration and muscle atrophy are the major pathological processes associated with ALS consistent with the role of nerve activity in muscle homeostasis and remodeling (Munson et al., 1997;Pette, 2001;Schiaffino et al., 2007). However, other cells may be involved in the pathogenesis of ALS and alteration in skeletal muscle homeostasis might represent one of the critical mediators of motor neuron degeneration. This hypothesis has been recently investigated by different groups, and we recently demonstrated that muscle selective expression of SOD1 mutation causes pathological alterations in skeletal muscle and induces pre-symptomatic sign of ALS at the level of spinal cord (Dobrowolny et al., 2008b).
In the present study, we extended our previous work and analyzed whether muscle-specific expression of SOD1 G93A is able to affect the expression of non-coding RNA and mRNA in the spinal cord of MLC/SOD1 G93A transgenic mice, compared with wild type littermates. Our findings are in agreement with previous studies showing that skeletal muscle is also a source of signals that influence neuron survival, axonal growth and maintenance of synaptic connections (Funakoshi et al., 1995;Dobrowolny et al., 2005). In the absence of trophic support, muscle can have a negative impact on the nervous system, and therefore, can contribute to the alteration in the functional connection between muscle and nerve.
The major findings of our study indicate that muscle-specific expression of SOD1 G93A can modulate relevant molecules of the genetic and epigenetic circuitry of the myelin homeostasis in the spinal cord of transgenic mice. In particular, we found down regulation of mir-1, mir-330, mir-29, mir-133, and mir-9 family members, whose dysregulation can have profound effects on neuronal physiology and pathology, including Huntington, Alzheimer, and Parkinson diseases (Saito and Saito, 2012). Although, it is known that deregulation of specific miRNAdependent regulatory circuitries correlates with the initiation and progression of several neurological disorders, the underlying mechanisms of these phenomena are still not fully understood.
Here we revealed the alteration of specific microRNAs and potential mRNA targets, which might be part of the short circuit that disrupts the communication between skeletal muscle and nerve and that might be part of the so called dying back phenomenon (Dadon-Nachum et al., 2011). In particular, among transcripts that are potential targets of the altered miRNAs, we revealed the presence of relevant markers of myelin homeostasis that are modulated by muscle restricted expression of SOD1 mutation inducing hypomyelination of the peripheral nerve.
The up-regulation of Pmp22 and Mpz proteins in the spinal cord of MLC/SOD1 G93A paralleled that of mRNA expression and supports the evidence that these factors are molecular targets of microRNAs, such as miR-1, miR-9, miR-133, and miR-330, that resulted differently modulated in the spinal cord of MLC/SOD1 G93A mice compared to wild type littermates. It has been demonstrated that Myelin protein zero is upregulated in oligodendroglia following axonal injury (da Silva et al., 2015) and Periaxin is expressed by myelinating Schwann cells in association with the dystroglycan complex through Drp2 (Sherman et al., 2012). Moreover, Pmp22 exhibits a dose dependent function as both overexpression and deletion result in hereditary neuropathy (Han et al., 2013). Dhh is essential for the structural and functional integrity of the peripheral nerve (Sharghi-Namini et al., 2006) and Osteglycine, Claudin -19 and Smoothelin together with Pmp22 and Prx are associated with reduced myelination of the spinal cord in the hypogravity motor syndrome (Chelyshev et al., 2014). Moreover, mutations in Mpz, Pmp22, Egr2, and Prx are associated with Charcot-Marie-Tooth neuropathy in which the over expression of these genes leads to defective myelination processes.
Regarding the mRNA-miRNA interplay, most of the putative interactions proposed here have not been previously described and deserve further investigation. Interestingly, the functional interaction of miR-9 with Peripheral myelin protein 22 (Pmp22) mRNA has been already demonstrated (Lau et al., 2008). In particular, miR-9 is down-regulated during oligodendrocyte differentiation and its expression level inversely correlates with the expression of its targets Pmp22. Moreover, it has been described that the inhibition of endogenous miR-29 overrides the miRNA-mediated repression of Pmp22 cultured Schwann cells during both development and post-crush injury (Verrier et al., 2009). Therefore, these data suggest that myelin gene expression is regulated by miRNAs.
Here we observed that the muscle specific expression of the SOD1 mutant gene induces the deregulation of both mir-9 and 29 together with myelin alteration in the sciatic nerve. These data have been validated by histological analysis of the sciatic nerve that shows a reduction in the myelin sheath in the MLC/SOD1 G93A mice model. In this regard it is interesting to note that the classical oligodendrocyte markers were downregulated (considering a fold change < 1.5 olig2 FC = −1.3 p = 0.02; Nkx2-2 FC = −1.36; p = 0.008; Mag FC = −1.39, p = 0.01). Therefore the reduction in oligodendrogenesis could be correlated with the hypomyelination in the MLC/SOD1 G93A mouse model.
In the spinal cord of SOD1 G93A transgenic mice that ubiquitously express the mutant SOD1 gene, the miRNA-9 expression is up-regulated (Zhou et al., 2013). In this model there is extensive degeneration of gray matter oligodendrocytes in the spinal cord prior to disease onset; actually new oligodendrocytes were formed but they failed to mature, resulting in progressive demyelination (Kang et al., 2013). The difference in the expression levels of miRNA-9 between the global SOD1 G93A and MLC/SOD1 G93A mice is likely due to SOD1 G93A gene ubiquitously expressed in all tissues, including muscle, motor neurons and glia. This might generate a synergistic toxic effect, leading to a more severe phenotype.
Although, most of the miRNA and mRNA inverted correlations proposed here have not been yet validated, the elevated number of potential interactions strongly points toward a role of skeletal muscle in myelin homeostasis. Nevertheless, although these results point toward some mechanisms of action of muscle specific expression of SOD1 G93A at the level of spinal cord, the elevated number of potential targets of the analyzed miRNAs make these mechanisms only mere suggestions. Additional studies will define which cell types, at the levels of spinal cord of MLC/SOD1 G93A mice, specifically exhibit changes in microRNAs and mRNAs.
Overall our study provides additional insights into the effects of muscle selective expression of SOD1 G93A on nerve homeostasis and reveals the potential miRNA and mRNA signature associated with the dying back phenomenon.


extfieldsofstudy: Biology Medicine

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Characteristics of dr1790 disruptant and its functional analysis in Deinococcus radiodurans

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.

Introduction
D. radiodurans exhibits resistance to the lethal and mutagenic effects of DNA damaging agents, including g-ray and UV radiation, hydrogen peroxide and desiccation (Battista, 1997;Makarova et al., 2001;Shu and Tian, 2010;Ghosal et al., 2005). These bacteria can survive~12 kGy g-ray irradiation, which generates approximately 200 double-strand and 3000 single-strand breaks per genome (Battista, 2000). The robustness of this bacterium reflects strong oxidative stress resistance mechanisms that protect proteins from oxidative damage (Wang et al., 1995;Markillie et al., 1999;Daly et al., 2007) and a DNA repair process that efficiently and precisely reassembles DNA fragments (Minton 1994;Slade et al., 2009). Antioxidant waste sites) and 30-50 mM Hg(II) and that effectively reduce Hg(II) to the less toxic volatile elemental Hg(0) (Brim et al., 2000). The cloning of toluene dioxygenase tod genes from Pseudomonas putida F1 into the chromosome of D. radiodurans conferred the ability to oxidize toluene, chlorobenzene, 3,4-dichloro-1-butene, and indole in a highly irradiating environment (Lange et al., 1998). The expression of IrrE, which is a global regulator for extreme radiation resistance in D. radiodurans, significantly enhanced salt tolerance in Brassica napus plants. Transgenic B. napus plants that express the IrrE can tolerate 350 mM NaCl, which is a concentration that inhibits the growth of almost all crop plants (Pan et al., 2009). The human bone marrow cell line KG1a, which was transformed with dr1709 from D. radiodurans, exhibited a much higher survival fraction than the original KG1a cells when treated with g-ray radiation (Shu and Tian, 2012). However, the underlying mechanisms of D. radiodurans resistance to stresses remain unclear. Therefore, the identification and functional analysis of new genes that are associated with anti-radiation, DNA repair and antioxidants will improve our understanding of the extreme radiation resistance mechanisms of this strain and provide strategies for research regarding the radiation damage defense and oxidative stress resistance systems of organisms.
dr1790, which is a gene that encodes a putative yellow-related protein homolog, was identified in the D. radiodurans genome (Makarova et al., 2001). Interestingly, this yellow-related protein is typically detected in insects and plays important roles in pigmentation and insect behaviors. The deletion of the yellow protein gene locus in Drosophila not only affects larval pigmentation but also appears to affect insect behavior (Maleszka and Kucharski, 2000;Drapeau et al., 2006). Yellow protein can be secreted from cells because this protein contains a secretion signal peptide (Drapeau, 2003). Furthermore, other members of the Yellow/Major Royal Jelly (MRJ) protein family are expressed in not only insects but also some bacterial and fungi species, suggesting that yellow proteins are evolutionarily ancient (Drapeau et al., 2006). Although a few studies have demonstrated an association between melanization and behavior in Drosophila, and a unique clade of genes from Apis mellifera may be involved in caste specification, the function of most yellow protein family members remains largely unknown (Ferguson et al., 2011). Currently, no studies concerning the function of yellow-related proteins in prokaryotes exist. DR1790 expression was induced in a D. radiodurans mutant strain that was deficient in OxyR, which is a peroxide sensor and transcription regulator that senses the presence of reactive oxygen species and that induces the antioxidant system of D. radiodurans . These findings prompted us to investigate the functions of this yellow-related protein homolog in this extremophilic bacterium.

Construction of mutant strains
The D. radiodurans strain R1Ddr1790 was constructed using a deletion replacement method as described previously . The primers that were used in this study are listed in Table 1. The primers p1 and p2 were used to amplify a BamHI fragment upstream of the targeted genes, and the primers p3 and p4 were used to amplify a HindIII fragment downstream of the targeted genes. The kanamycin resistance cassette containing the GroEL promoter was obtained from the pRADK shuttle plasmid (Gao et al., 2005). The three DNA fragments were digested and ligated; then, the ligation products were used as templates for PCR (30 cycles at 94°C for 1 min, 55°C for 45 s, and 72°C for 1 min) with p1 and p4. The resulting PCR fragments were transformed into D. radiodurans cells using the CaCl 2 technique, and the mutant strains were selected on TGY agar plates supplemented with 20 mg/mL kanamycin.

Complementation of R1Ddr1790
The complementation plasmid was constructed as described previously (Gao et al., 2005;Wu et al., 2009). Briefly, chromosomal DNA was isolated from wild type strains. The 1167-bp region containing the dr1790 gene was PCR-amplified (35 cycles at 94°C for 1 min, 58°C for 50 s and 72°C for 1 min) using the primers DR1790 com F and DR1790 com R (Table 1) and ligated into the pMD18 T-Easy vector (Takara, JP); the resulting construct was designated as pMD-dr1790. After digestion with NdeI and BamHI, the target gene dr1790 was ligated into NdeIand BamHI-pre-digested pRADK, and the resulting construct was designated as pRAD-dr1790. The complementation plasmids were confirmed by PCR and DNA sequence analyses; thus, transformation into R1Ddr1790 generated the functional complementation strain mutant Dr1790com.

Measurement of growth rate
The growth rate was measured as described previously (Mattimore et al., 1995). Briefly, 500 mL overnight culture of each strain was transferred to 50 mL TGY medium. The culture was grown at 30°C with agitation (200 rpm). Then, the culture dilutions were spread onto TGY agar plates after 2 (t 1 ) and 4 h (t 2 ). The plates were incubated at 30°C for 5 days, and subsequently, the number of colony-forming units (CFU) was determined. The dou-ble time (g) was calculated according to the following formula: g = ln2 / ((log 10 N 2 -log 10 N 1 ) 2.303/Dt), where N 1 is CFU per milliliter at t 1 , and N 2 is CFU per milliliter at t 2 . dr1790 deleted mutant in DR 603

Cell survival under oxidative stress and ionizing radiation
The hydrogen peroxide sensitivity of D. radiodurans cells was assayed as described previously , with some modifications. The cells were harvested during the early stationary phase (OD 600nm = 1.0), washed twice and re-suspended with phosphate buffer (20 mM, pH 7.4). An aliquot was removed as a control, and the remaining suspension was treated with hydrogen peroxide at a final concentration of 20 mM. The mixture was incubated at 30°C in an orbital shaker. Catalase (Sigma-Aldrich) was added in excess (15 U) to terminate the H 2 O 2 treatment. Then, the cells were diluted and spread onto solid TGY media to determine the number of CFUs. The survival fractions were defined as a percentage of the CFU obtained in the treated sample compared with the control. The data are presented as the means ± SD of three independent experiments.
The cell survival fractions under ionizing radiation were determined using a previously described method .

Membrane localization of the DR1790 protein
The plasmid pRADG-dr1790 was constructed as described previously (Gao et al., 2008). pRADG-dr1790 was transformed into the R1Ddr1790 mutant strain. The transformant was obtained by chloramphenicol-resistance selection. The transformant was grown to the exponential phase (OD 600nm is approximately 0.8), spread on a glass slide and examined using a laser confocal microscope (Zeiss LSM510, Germany).

Membrane integrity assessment
Differences in membrane permeability between the varying strains were assessed using a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA). This system employs two nucleic acid stains: green-fluorescent SYTO9 stain and red-fluorescent propidium iodide (PI) stain. Live cells with intact membranes fluoresced green, while dead cells or cells with compromised membranes fluoresced red. Bacterial cells were grown to mid-exponential phase, and a 1-mL aliquot of the culture was normalized to an OD 600nm equal to 0.6, washed twice with PBS, and resuspended in 1 mL PBS. The bacterial suspensions were stained with the nucleic acid dyes according to the manufacturer's protocol; then, 10 mL stained bacteria was spotted onto glass coverslips and visualized using a Leica DM4000B wide-field epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). In total, 10 different fields were viewed for each strain, and the numbers of green, red or mixed cells were counted for each field.

Transcriptome analysis
The procedures used for microarray hybridization and data analyses were performed as described previously . Briefly, total RNA was prepared from three replicates of wild type and R1Ddr1790 mutant cells. Approximately 16 mg total RNA was annealed with 10 mg random hexamer primers in a total volume of 20 mL at 70°C for 10 min, followed by incubation on ice for 2 min. cDNA synthesis was performed at 42°C overnight in a 31-mL reaction mixture using SuperScript III Reverse Transcriptase (Invitrogen) with 0.5 mM dNTP mix containing amino allyl-dUTP (GE, Piscataway, NJ, USA). The reaction was terminated by adding 20 mL 0.5 M EDTA and 20 mL 1 M NaOH, followed by heating at 65°C for 20 min. The reaction mixture was neutralized with 50 mL 1 M HEPES buffer (pH 7.0), and unincorporated amino allyl-dUTPs were removed by ultra-filtration using YM 30 columns (Millipore). The cDNA was coupled to 1 pmol Cy3 or Cy5 dye (GE) in 0.1 M sodium carbonate buffer for 2 h at room temperature, and free Cy3 or Cy5 was removed. The labeled pools of wild type and R1Ddr1790 mutant cDNA were mixed and simultaneously hybridized with the DNA chips in a solution containing 3X saline sodium citrate (SSC), 0.3% SDS, and 24 mg unlabeled herring sperm DNA (Gibco BRL, Gaithersburg, MD, USA). Normalization and statistical analysis were performed in the R computing environment (version 2.2.0, R with Aqua for Windows) using the linear modeling features of the Limma microarray data package (Wettenhall et al., 2004). Before channel normalization, microarray outputs were filtered using Limma to remove spots with poor signal quality by excluding data points with a mean intensity less than two standard deviations above the background in both channels. Then, global LOESS normalization was used to normalize all data, and three replicate spots per gene in each array were used to maximize the robustness of the differential expression measurement of each gene via the "lmFit" function. The transcriptome analysis data were deposited in the Gene Expression Omnibus database under accession no. GSE22628.

Real-time quantitative PCR
The genes of interest were identified using real-time quantitative PCR to validate the results of the microarray data. dr0089, which is a gene whose expression is unaffected by H 2 O 2 and ionizing radiation, was used for normalization. Briefly, first-strand cDNA synthesis was performed in 20-mL reactions containing 1 mg each DNase I-treated and purified total RNA sample and 3 mg random hexamers. The real-time PCR amplification was performed 604 Cheng et al.
using a Toyobo SYBR Green I Real Time PCR kit (Japan) according to the manufacturer's instructions under the following conditions: 94°C for 2 min, followed by 40 cycles at 94°C for 10 s, annealing at 56-62°C for 15 s and 72°C for 30 s. All assays were performed using a Stratagene Mx3005P qPCR system (Stratagene, Cedar Creek, TX, USA).

Proteomics analysis
Proteomic analysis of the mutant compared with the wild type strain was performed using 2-DE and data analyses, in-gel digestion, MALDI-TOF MS analysis and a PMF spectra-based database search (Lu et al., 2009).

Growth of the deletion mutant
We assayed the doubling time of cells in the lag and log phases. The R1Ddr1790 mutant doubling time (2.1 ± 0.4 h) was not significantly slower than the doubling time (1.5 ± 0.4 h) of the wild type R1 strain under aerobic conditions during the lag phase (p > 0.05) ( Figure 1A). However, the R1Ddr1790 mutant doubling time (3.1 ± 0.5 h) was slightly slower than the doubling time (1.6 ± 0.2 h) of the wild type R1 strain under aerobic conditions during the log phase (p < 0.05) ( Figure 1B).

The deletion mutant was sensitive to oxidative stress and radiation
The yellow-related protein homolog DR1790 from D. radiodurans functions has been implicated in cell resistance to oxidative stress and radiation. The R1Ddr1790 mutant was sensitive to hydrogen peroxide treatment and g-ray radiation. Compared with the wild type strains, the survival of the R1Ddr1790 mutant cells decreased nearly 15-fold in response to 30 mM hydrogen peroxide ( Figure 2A) and nearly 3-fold in response to a 8 kGy dose ( Figure 2B).
To determine whether the loss of DNA damage tolerance in the R1Ddr1790 mutant reflected the absence of dr1790 and not a polar effect of this mutation, the wild type allele of this gene was cloned into pRADgro, which is a Deinococcus expression vector, and the protein was expressed in R1Ddr1790 mutant cells. The radiation and oxidative resistance of the complemented mutant Dr1790com strain nearly recovered to the phenotype of the wild type strain (Figure 2), suggesting that the sensitivity of the R1Ddr1790 mutant reflected the absence of the dr1790 gene.
Comparison of intracellular protein oxidation levels between the wild type R1 strain and the R1Ddr1790 mutant strain The level of protein oxidation in the R1Ddr1790 mutant was analyzed and compared with that of the wild type R1 strain (Figure 3). The total protein carbonyl contents measured in the wild type and mutant strains were 0.012 and 0.015 mmol/mg, respectively, indicating that the mutant strain exhibited relatively higher levels of protein oxidation compared with the wild type strain in the absence of oxygen stress. Following H 2 O 2 treatment, intracellular protein carbonylation significantly increased in both the wild type and R1Ddr1790 mutant strains. The carbonyl content in the R1Ddr1790 mutant post-H 2 O 2 treatment was 0.023 mmol/mg protein, which was significantly higher than the content in the wild type cells (0.017 mmol/mg protein, p < 0.05), suggesting that the intracellular proteins in the mutant cells lacking DR1790 were more sensitive to oxidative damage than those in the wild type cells.

Membrane localization of the DR1790 protein and membrane integrity of the R1Ddr1790 mutant strain
Fusion gene expression of the green fluorescence protein (eGFP) gene and dr1790 was performed and analyzed by fluorescence microscopy to confirm the localization of the DR1790 protein (Gao et al., 2008). Figure 4 shows that eGFP-labeled protein (green fluorescence) was localized to the cell membrane; the yellow fluorescence displayed in the merged picture indicates the co-localization of eGFPlabeled proteins and FM4-64 (red fluorescence)-labeled membranes, confirming that DR1790 is a membrane protein.
The membrane integrity of the mutant strain was analyzed based on permeability assays using membrane-permeant and membrane-impermeant fluorescence-labeled nucleic acids. The R1Ddr1790 mutant incorporated both the membrane-impermeant dye propidium iodide (PI) and the membrane-permeant dye SYTO9 ( Figure 5). Of 1384 mutant bacterial cells counted in 10 different fields, 20% of the cells incorporated PI (red). In contrast, wild type R1 and complemented mutant Dr1790com strains incorporated SYTO9 (green); however, only 1% of the cells were PIpositive among the 1464 wild type and 1538 complemented mutant bacteria that were counted in 10 independent fields ( Figure 5). Thus, the R1Ddr1790 mutant showed a high proportion of damaged membranes (20% red cells observed in the mutant field) compared with wild type and comple- 606 Cheng et al.    mented mutant strains. This result suggests that the DR1790 protein contributes to membrane permeability.
Transcriptional and translational profiles of the DR1790 mutant vs. the wild type strain 2-DE and MS analyses were applied to compare the differential protein expression profiles of the R1Ddr1790 mutant and the wild type R1 strains ( Figure 6). Ten protein spots showing two-fold changes in intensity in the R1Ddr1790 mutant compared with the wild type R1 were observed, including growth-related metabolism enzymes (IDH, MDH and FBP2), the predicted transmembrane protein transporter DR1909, and the chaperone protein DnaJ ( Table 2). The limited information acquired by 2-DE analysis prompted the use of DNA microarray analysis to investigate this issue further.
The transcriptome of the R1Ddr1790 mutant was analyzed and compared with that of the wild type strain under normal growth conditions using oligonucleotide microarray to examine the expression of the entire gene repertoire of D. radiodurans in response to dr1790 knockout. In the present study, a two-fold difference in the relative transcription level was selected as the threshold for microarray data analysis as described previously by Chen et al. (2008). We observed that 1.5% of the genes represented on the microarray (n = 46) were differentially transcribed in the R1Ddr1790 mutant compared with the WT. Among these genes, 27 were up-regulated (Table 3), and 19 were downregulated (Table 3). These genes were involved in DNA/RNA repair, energy metabolism, various transporters, proteases and chaperones, stress responses, and translation and transcription functions.
Among the up-regulated genes in the R1Ddr1790 mutant, three genes were categorized as proteinase genes, six genes were related to protein quality control, and some genes encoded unknown/hypothetical proteins. Similarly, the down-regulated genes in the R1Ddr1790 mutant included four genes that were related to secreted proteins. The effectiveness of the microarray data was further confirmed by real-time quantitative RT-PCR (Table 4). Nota-dr1790 deleted mutant in DR 607   Cheng et al. bly, many molecular chaperones and proteinases were positively regulated in the R1Ddr1790 mutant, and transporters and kinases were negatively regulated in the R1Ddr1790 mutant. These data demonstrate that the deletion of the dr1790 gene significantly increased the amount of misfolded proteins in the cell. Some secreted proteins and transmembrane protein transporters were repressed, indicating that the DR1790 protein could be associated with secretory factors in the membrane.

Discussion
The extreme resilience of D. radiodurans to oxidative and radiation stresses is imparted synergistically by the efficient protection of proteins against oxidative stress and efficient DNA repair mechanisms, enhanced by functional redundancies in both systems (Slade and Radman, 2011). Maleszka et al. (2000) identified an orphan protein (DR1790) in D. radiodurans belonging to the yellowrelated protein family, which was originally identified in Drosophila. A mutation in the yellow-related protein in Drosophila affects the pigmentation of larvae and exerts some effects on insect behavior (Drapeau et al., 2006). In the present study, the predicted yellow-related protein DR1790, which belongs to the ancient Yellow/MRJ protein family, was confirmed to be a membrane-binding protein.
A null-mutant strain (R1Ddr1790) exhibited reduced survival after gamma irradiation and H 2 O 2 treatment, demonstrating that DR1790 is involved in the radioresistance and antioxidant mechanisms of D. radiodurans. Protein, rather than DNA, was suggested to be the principal target of radiation and free radicals, and the degree of cell resistance was determined based on the level of oxidative protein damage (Daly et al., 2007). We observed that the total protein car-bonyl contents increased in the R1Ddr1790 mutant under normal conditions and H 2 O 2 treatment, demonstrating that the absence of DR1790 increased oxidative damage in cells.
Cellular membranes, which are composed of lipids, proteins, and carbohydrates, are damaged by radiation. The melting of membranes under stress results in permeability barrier loss and leakage, as well as the inability to maintain a proton gradient for respiration. The D. radiodurans cell envelope consists of at least five layers (Lancy et al., 1978). D. radiodurans irradiated with 4 kGy loses up to 30% wet weight resulting from the loss of polysaccharides into the growth medium, which suggests permeability alterations in the cell envelope (Mitchel, 1976). For retaining membrane integrity, D. radiodurans cells were much more resistant to high temperatures when exposed in the dried state as opposed to cells in suspension (Bauermeister et al., 2012). The R1Ddr1790 mutant showed a high proportion of damaged membranes (20% red cells observed in the mutant field) compared with wild type and complemented mutant strains. This result suggests that the DR1790 protein contributes to membrane permeability. Consequently, the mutant strain was more sensitive to both ionizing radiation and oxidative stress. However, how DR1790 contributes to bacterial membrane integrity remains unclear. DR1790 may be required for the stability of membrane protein complexes to restore the osmotic imbalance rapidly, and the absence of DR1790 may result in less stability or improperly gated channels or pores. Thus, the isolation of the protein partners of DR1790 may help to clarify the role of this protein in membrane homeostasis. Alterations in membrane integrity may also contribute to the increased sensitivity of R1Ddr1790 mutants to oxidative and radiation stresses.  In the present study, some genes that are involved in protein quality control, such as dr1114 (HSP20), dr0129 (dnaK), dr0126 (dnaJ), dr0607 (groEL), dr1046 (ATP-binding subunit ClpB), and dr1974 (ATP-dependent protease LA, Lon1), were strongly induced in R1Ddr1790 mutants. The induction of these chaperones and proteases suggested that many damaged proteins aggregated in the R1Ddr1790 mutant. Chaperones assist in non-covalent folding or unfolding and in the assembly or disassembly of protein structures in the cell, but do not occur in these structures during the performance of normal biological functions after having completed folding and/or assembly. DnaK/DnaJ/GrpE and GroEL/ES are the two primary chaperone foldase systems in prokaryotic cells (Hoffmann et al., 2004). ATP-dependent proteases function in protein processing and play an essential role in diverse stress responses (Gottesman, 2003). In D. radiodurans, the majority of cellular proteolysis is performed by ATP-dependent proteases that belong to the Lon (Lon1 and Lon2) and Clp families (ClpA, ClpB, ClpC, ClpX and ClpP). The ClpPX protease is required for radioresistance and regulates cell division after g-irradiation in D. Radiodurans (Servant et al., 2007). ClpB from Myxococcus xanthus functions as a chaperone protein and plays an important role in cellular heat and osmotic stress tolerance mechanisms during both vegetative growth and development (Pan et al., 2012). ClpB and the DnaK system act synergistically to remodel proteins and to dissolve aggregates (Doyle et al., 2007). HSPs function as molecular chaperones that prevent protein denaturation and aggregation (Feder and Hofmann, 1999;Matuszewska et al., 2008). Additionally, some genes that are involved in protein secretion and transport are strongly suppressed in R1Ddr1790 mutants, such as secreted proteins (DR1900, DR1901, DRA0300, and DR2544) and transmembrane transporter proteins (DR1909), indicating that the DR1790 protein could be related to secretory factors in the membrane. D. radiodurans contains many secreted proteases and transporters that provide exogenous amino acids as protein building blocks and peptides as components of manganese complexes (Slade and Radman, 2011). After irradiation in D. radiodurans, 10 secreted subtilisin-like proteases, and 4 peptide and amino acid ABC transporters were highly induced (Makarova et al., 2000;Ghosal et al., 2005). Thus, the low growth rate and sensitivity to hydrogen peroxide and radiation in the R1Ddr1790 mutant were closely associated with the induction of these chaperones and proteases and with the suppression of secreted and transported proteins. Additionally, the expression of some genes involved in antioxidant systems, electron transport, and energy metabolism were also significantly altered by the disruption of DR1790.
In conclusion, we presented the first experimental evidence that a protein from the ancient yellow protein family plays a role in the survival of prokaryote cells during a damage response. The DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses. Further studies are required to understand the mechanisms of the action that are mediated by DR1790 during this process and to identify critical protein interactions.