BACKGROUND
alzheimer’s disease  is the most common neurodegenerative disease worldwide and is characterized by cognitive dysfunction and neuronal degeneration  <cit> .
several pathological markers define ad, including altered amyloid beta  production, aβ aggregation, hyperphosphorylated tau, neurofibrillary tangles  and senile plaques  <cit> .
microtubule-associated protein tau has been implicated in tangles and malfunction of cytoskeleton-related trafficking  <cit> .
aβ derived from amyloid precursor protein  is involved in up-regulation of protein aggregates and related dysfunction in app processing resulting from mutations in app, presenillin <dig>  or presenillin <dig>  for early-onset ad and apolipoprotein e  for late-onset ad  <cit> .
omics approaches based on microarray and next generation sequencing  technologies have been used to study the pathological mechanism of ad  <cit> .
genome-wide integrative approaches identify functional drivers of the disease and predict their specific regulatory functions.
using anti-correlation or target prediction programs, many studies have focused on the prediction of target gene or mirna function; however, this approach has drawbacks because it may deliver false positive interactions and exhibit high noise ratios  <cit> .
in this study, we hypothesized that simultaneous genome-wide analysis of mrna and mirna expression in an ad model at different disease stages will reveal differential gene expression patterns in ad, which may help to predict stage-dependent gene markers in ad pathology.
to this end, we used tg <dig> transgenic mice, which contain mutant human app  with swedish , florida , and london  familial alzheimer’s disease  mutations, as well as human ps <dig> with m146l and l286v fad mutations  <cit> .
this ad transgenic model has been reported to show ad-like phenotypes such as rapid accumulation of aβ, reduced levels of synaptic marker protein expression, increased p <dig> levels, neuronal loss, and memory impairment  <cit> .
RESULTS
module prediction for ad
using microarray data, we identified three modules that were closely related with ad.
the function of each module was identified by the go enrichment test of david.
the mirna-mrna interactions in ad were ranked by their topological importance through our co-expression network analysis and the ranking scores were applied to the pagerank algorithm.
in total,  <dig>  differentially expressed genes  were identified and  <dig> modules were predicted from the expression microarray data.
modules and maturation factors were predicted using the pearson correlation method .
the significance of each module was calculated using the wgcna package based on gene significance  and module membership .
three modules showed positive correlations with ad .figure  <dig>
gene significance scatter plot for several gene modules.
left panel represents gene scatter plot for the modules with genetic correlations and right panel represents the modules with age-related correlations.
the statistically significant relationship between gene expression and genetic mutation or age was evaluated by pearson correlation.
the green module showed positive correlation  with ad for sodium ion transport .
ad has been reported to be associated with impaired glutamate clearance and depressed na+/k+ atpase levels in the brain  <cit> .
several solute carrier family members were found in the green module .
the pink module showed correlations with ad  and disease stage .
genes related to ion homeostasis and immune responses were identified in the pink module .
metal ion dyshomeostasis is a well-recognized cofactor in several neurodegenerative disorders  <cit> .
in ad, the process of aβ misfolding and plaque aggregation is greatly influenced by alterations in the homeostasis of the aforementioned metal ions  <cit> .
a strong genetic association between aβ formation and ad has been reported in several studies  <cit>  and we found that aβ is highly expressed in the ad mouse model used in this study.
the yellow module was correlated with ad  and included genes related to cell death, apoptosis, and development .
ad is correlated with region-specific neuronal cell loss  <cit> .
notch  <dig>  found in the yellow module, is known to regulate the notch signaling pathway, which is involved in controlling the fate of neural and non-neural cells in development process and adults  <cit> .
several observations suggest that enhanced notch signaling and expression could be instrumental in neurodegeneration in ad  <cit> .
huntingtin interacting protein  <dig> , found in the yellow module, is related to cell death through caspase- <dig> activation in immortalized hippocampal neuroprogenitor cells  <cit> .
thus, the functions of the proteins in the green, pink, and yellow modules are directly associated with ad.
the black, brown and red modules showed relationships with disease stage and are involved in brain development including central nervous system development, neurogenesis, and forebrain development .
the blue and turquoise modules showed significant relationships with ad and disease stage.
the gene list for each of the  <dig> modules and the gsa results are presented in additional file 1: table s <dig> and additional file 2: table s <dig>
mirna-mrna regulatory network in tg <dig> mice
we constructed a co-expression network from the functional gene module to study mirna-mrna interactions and the regulation of gene expression by mirna.
we integrated several approaches to predict mirna targets and module regulators, which were predicted using 3-target site analysis.
although black, brown, and yellow modules did not show direct correlations with genetic ad , these  <dig> modules were considered as ad related mirna modules because dysregulation of neurogenesis often cause neurodegenerative diseases.
using all these  <dig> modules , we calculated all the pair expression correlations from target relations using sequence-based target prediction results and selected  <dig> mirna-mrna interactions by anti-correlation.
the constructed co-expression weighted network  contains the black, brown, red, and pink modules.
mrnas in the red module correlate significantly with disease stage .
mir-34b, which interacts with genes in the brown and pink modules, has been proposed as a key regulator to integrate age-associated diseases in the vertebrate brain .
it closely interacted with mrnas in the brown and pink modules.
some genes in the disease stage-related modules have been reported to be associated with ad.
for example, the members of the black and red modules are related to neurogenesis in normal developmental stages.
the ad network was constructed using the yellow and green modules .
in particular, mir- <dig> and mir- <dig> are known to be down-regulated in ad patients  <cit> .figure  <dig>
the subnetwork of weighted coexpression networks of mirna-mrna in ad.
the subnetwork of brown, grey, and pink module genes and related mirnas.
as presented in figure  <dig> transgenic plots , these  <dig> modules show negative correlations.
the subnetwork of green  and yellow  module genes and related mirnas.
the subnetwork of weighted coexpression networks.
a compact network was constructed from arrestin domain containing  <dig>  neighbors and related mirnas to their target genes.
the interaction between mirna and their target genes were predicted by sequence analysis and expression anti-correlation.
significant interactions of mirna-mrna were predicted by the co-expression network.
we assigned priority to mirna-mrna targets based on network topology.
we assumed that highly ranked genes in mirna-mrna networks are more likely to play critical roles in the pathophysiology of ad.
using the pagerank algorithm, which is useful for describing the relative node importance in a network connection, we measured the pagerank score for mirna-mrna interactions and assigned the rank of interaction priority using only differentially expressed mirnas  and deg.
we determined that  <dig> mirna-mrna interactions were significant targets and confirmed  <dig> mirnas with a high pagerank score of < <dig>  .
microglia induction in ad can be explained by our mirna-mrna interaction prediction system.
our results predicted that mir-34b was involved in the regulation of colony stimulating factor  <dig> receptor , which has been reported to be critical for survival and proliferation of microglia  <cit> .
our microarray data showed that csf1r was significantly down-regulated.
aging and neurodegeneration seem to be modulated by mir- <dig> expression through microglial activation  <cit>  and activation of microglial chemokine receptor  <dig>  is known to lead to neuronal death in ad.
our current microarray data demonstrate that expression of chemokine ligand  <dig>  and adam metallopeptidase domain  <dig>  increased in tg <dig> mice at 8 months of age.
adam <dig> is a predicted target of mir- <dig> and mir- <dig> and is involved in cx3cl <dig> cleavage.
cleavage of cx3cl <dig> by adam <dig> can lead to microglial activation of neuronal degeneration  <cit> .
differential expression of mirna in tg <dig> mice
in total,  <dig> differentially expressed  mirnas were identified in the early- and late-stage ad mouse model .
we identified several mirnas that correlated with ad .
for example, mir- <dig>  which is down-regulated in the brains of ad mice, is specifically expressed in the brain and has been demonstrated to negatively regulate dendritic spine formation in vitro
<cit> .
mir- <dig> is enriched at synapses and modulate synaptic development and spine size through the control of acyl-protein thioesterase- <dig>   <cit>  and the depalmitoylation of g protein alpha  <dig> , a down-stream target of apt <dig>  which is an activator of rho down-stream of g-protein coupled receptor   <cit> .
mir-146b is down-regulated in ad mice and has been reported to be consistently altered in both hippocampus and medial frontal gyrus in ad mouse models  <cit> .
mir-146b could be inferred as a negative regulator of innate immunity and its down-regulation in the ad brain provides support for an induction of toll like receptor  signaling in ad  <cit> .
mir- <dig> is highly expressed in the cortex during early fetal development and is related to the growth spurt in the cerebellum compared to the cortex  <cit> .
mir-26a was observed to be down-regulated in tg <dig> mice at  <dig> months of age.
mir-26a has been reported to be down-regulated in nasopharyngeal carcinomas and to suppress cell proliferation and colony formation by inducing g1-phase cell-cycle arrest  <cit> .
mir-34b and mir-34c were up-regulated in our ad model mice and have been reported to be up-regulated in ad patients  <cit>  .
we validated the expression levels of three mirnas  from the list of candidates.
real time-pcr results showed that at  <dig> months of age, mir139-5p expression showed tendency to increase , in the hippocampi of tg <dig> mice compared to littermate controls .
the expression of mir340-5p and mir3470a did not increase significantly in mutant tg <dig> mice at either  <dig> or  <dig> months of age.figure  <dig>
verification of the expression of mrna and mirna in the hippocampus of tg <dig> mice.
the mrna levels of arrdc <dig>  protein phosphatase  <dig> regulatory subunit 3c  and splicing factor, proline- and glutamine-rich  in the hippocampus of tg <dig> mice were verified using rt-pcr.
or mutant tg <dig>  mice; §: p <  <dig>  between lm or mt mice; §§: p <  <dig>  between lm or mt mice).
the expression levels of mir139-5p, mir340-5p, and mir3470a in the hippocampus of lm or mt mice were determined using real-time pcr.
differential expression of mrna in tg <dig> mice: arrdc <dig>  ppp1r3c, sfpq and metabolic dysfunction
using integration analysis, we predicted functions of mrna and mirna associations in ad pathology.
focusing on that many neurodegenerative diseases are known to be caused by metabolic dysfunction, we decided to validate our prediction system through bio-energy metabolism related genes such as arrestin domain containing  <dig> , a predicted target of mir- <dig>  mir- <dig>  mir- <dig> and mir-3470a, in the brown module .
to validate the network topology analysis system, we selected significant interactions in non-demir and determined the target gene expression.
the mrna level of arrdc <dig> was increased in the hippocampus of tg <dig>  mice at 4 months of age compared to their littermate  controls.
the mrna level of arrdc <dig> was significantly increased in an age-dependent manner in the hippocampus of lm controls .
arrdc <dig> mrna also showed an age-dependent increase in mt mice.
interestingly, we determined that protein phosphatase  <dig> regulatory subunit 3c  exhibited direct relationships with age or ad mutation in consequence map data .
the mrna levels of ppp1r3c increased with age in lm controls and had a tendency to increase in mt mice at 4 months of age compared to lm controls .
we determined that mrna levels of ppp1r3c and splicing factor proline and glutamate-rich  decreased with age in mt mice and also decreased in mt mice at the late symptomatic stage compared to lm controls .
we next determined arrdc <dig> and sfpq protein expression levels by performing semi-quantitative western blot analysis using the hippocampus samples from mt mice and lm controls at 10 months of age.
the protein expression levels of arrdc <dig> and sfpq decreased in mt mice compared to lm controls .
co-immunoprecipitation data showed direct molecular interaction between arrdc <dig> and sfpq proteins and this interaction was increased in mt mice compared to lm controls .figure  <dig>
molecular interaction between arrdc <dig> and sfpq proteins.
the protein levels of arrdc <dig> and sfpq in the hippocampus of tg <dig> mice were determined using western blot.
the molecular interaction between arrdc <dig> and sfpq proteins was detected by co-immunoprecipitation experiment.
arrdc <dig> has been implicated in the regulation of body mass and energy metabolism and plays a role as a tumor suppressor .
as a down-stream target of arrdc <dig>  sfpq has been implicated in cellular processes such as transcriptional regulation and rna splicing, both of which are related to circadian rhythms.
sfpq is expressed in neurons and glia and is confined to the nuclei in both cell types  <cit> .
ppp1r3c could be induced by hypoxia as a hypoxia induced factor -targeted gene.
protein phosphatase  <dig>  is known to target glycogen  and to regulate glycogen metabolism  <cit> .
arrdc <dig>  ppp1r3c, and sfpq belong to a metabolism-related category of genes, as we demonstrated in the brown module.
previously, poll et al.
demonstrated that arrestin-dependent gpcr signaling is controlled by rapid dephosphorylation of pp <dig>  <cit> .
for functional validation of arrdc <dig>  we overexpressed arrdc <dig> by transfection or silenced arrdc <dig> expression with sirna in primary cultured cortical neurons from tg <dig>  mice and littermate  controls.
arrdc <dig> overexpression increased the mrna levels of arrdc <dig>  ppp1r3c, and sfpq in the cortical neurons of both lm and mt mice .
after silencing with arrdc <dig> sirna, the mrna levels of ppp1r3c and sfpq decreased in mt cortical neurons .
real time-pcr results showed that mir139-5p and mir3470a expressions have tendency to decrease after arrdc <dig> overexpression in both lm and mt primary cortical neurons .
we also determined the length and the number of neurites in primary cultured neurons from tg <dig> mice using anti-tau antibody.
the number of tau-positive neurons from mt mice significantly decreased and the length of neurites decreased as well compared to lm primary neurons.
however, arrdc <dig> overexpression could not alter the length or the number of neurites compared to vehicle treated mt neurons .
to determine arrdc <dig>  sfpq, and ppp1r3c mrna levels and mirna interaction in aβ toxic condition, we administered 2 μm aβ into the primary cultured cortical neurons from tg <dig> mice.
after aβ treatment, the mrna levels of arrdc <dig>  ppp1r3c, and sfpq increased in the cortical neurons of both lm and mt mice .
real time-pcr results showed that after aβ treatment, mir139-5p and mir3470a expressions have tendency to increase in cortical neurons of tg <dig> mice compared to vehicle treated mt neurons .
to detect luciferase activity of arrdc <dig> promoter in toxic condition, we transfected arrdc <dig> promoter into primary cultured cortical neurons of tg <dig> mice then administered aβ.
luciferase activity for arrdc <dig> promoter significantly increased in mt cortical neurons compared to lm control .
also after aβ treatment, luciferase activity for arrdc <dig> promoter significantly increased in lm cortical neurons compared to vehicle treatment .figure  <dig>
validation of arrdc <dig> function in primary cultured cortical neurons from tg <dig> transgenic mice.
overexpression of arrdc <dig> gene in primary cultured cortical neurons from tg <dig> mice.
rt-pcr results show that the mrna levels of ppp1r3c and sfpq were increased by arrdc <dig> overexpression.
knock-down of arrdc <dig> gene in primary cultured cortical neurons from tg <dig> mice.
the mrna levels of ppp1r3c and sfpq were decreased by the treatment of arrdc <dig> sirna.
real-time pcr data show that the expression levels of mir139-5p and mir3470a had decreasing tendency in the primary cortical neurons of tg <dig> mice.
immunocytochemical staining show no significant enhancement in the number and length of neurites in arrdc <dig> overexpressed primary cortical neurons from tg <dig> mice .
determination of the expression of arrdc <dig> related mrna/mirna under a administration.
rt-pcr show that the mrna levels of arrdc <dig>  ppp1r3c and sfpq were increased by aβ administration in primary cultured cortical neurons of tg <dig> mice.
real time pcr show that the expression levels of mir139-5p and mir3470a were not significantly altered by aβ administration in primary cortical neurons from tg <dig> mice.
luciferase activity of arrdc <dig> promoter increased after aβ treatment in lm cortical neurons but not in mt neurons.
taken together, these data suggest that arrdc <dig>  ppp1r3c, and sfpq interact with each other in regulating the age- and disease-specific gene expression of similar biological pathways such as bioenergetics and metabolism, which are very strong candidate categories implicated in the pathophysiological progressive changes of neurodegeneration .
CONCLUSIONS
our results demonstrate ad-specific changes in the mirna regulatory system and a relationship between the levels of mirnas and their targets in the ad hippocampus.
we identified ad-related modules and provided a specific network that integrates differential mirna and mrna expression profiles.
we proposed relevant mirna-mrna target relationships in an ad based on network topological priorities and mirna mediated mechanism in ad pathology.
our data highlight the functions and mechanisms of various mirnas and their target genes in the molecular pathology of ad including the genes which are related in ion transport, ion homeostasis, immune responses, cell death, apoptosis, development, and bio-energy metabolism.
knowledge of candidate genes identified through the simultaneous genome-wide profiling of mirna and mrna may be useful for the development of future therapeutics in ad.

 alzheimer’s disease is the most common neurodegenerative disease worldwide and is characterized by cognitive dysfunction and neuronal degeneration. several pathological markers define ad, including altered amyloid beta production, aβ aggregation, hyperphosphorylated tau, neurofibrillary tangles and senile plaques. microtubule-associated protein tau has been implicated in tangles and malfunction of cytoskeleton-related trafficking. aβ derived from amyloid precursor protein is involved in up-regulation of protein aggregates and related dysfunction in app processing resulting from mutations in app, presenillin or presenillin for early-onset ad and apolipoprotein e for late-onset ad. omics approaches based on microarray and next generation sequencing technologies have been used to study the pathological mechanism of ad. genome-wide integrative approaches identify functional drivers of the disease and predict their specific regulatory functions. using anti-correlation or target prediction programs, many studies have focused on the prediction of target gene or mirna function; however, this approach has drawbacks because it may deliver false positive interactions and exhibit high noise ratios. in this study, we hypothesized that simultaneous genome-wide analysis of mrna and mirna expression in an ad model at different disease stages will reveal differential gene expression patterns in ad, which may help to predict stage-dependent gene markers in ad pathology. to this end, we used tg transgenic mice, which contain mutant human app with swedish, florida, and london familial alzheimer’s disease mutations, as well as human ps with m146l and l286v fad mutations. this ad transgenic model has been reported to show ad-like phenotypes such as rapid accumulation of aβ, reduced levels of synaptic marker protein expression, increased p levels, neuronal loss, and memory impairment.  module prediction for ad using microarray data, we identified three modules that were closely related with ad. the function of each module was identified by the go enrichment test of david. the mirna-mrna interactions in ad were ranked by their topological importance through our co-expression network analysis and the ranking scores were applied to the pagerank algorithm. in total, differentially expressed genes were identified and modules were predicted from the expression microarray data. modules and maturation factors were predicted using the pearson correlation method. the significance of each module was calculated using the wgcna package based on gene significance and module membership. three modules showed positive correlations with ad.figure gene significance scatter plot for several gene modules. left panel represents gene scatter plot for the modules with genetic correlations and right panel represents the modules with age-related correlations. the statistically significant relationship between gene expression and genetic mutation or age was evaluated by pearson correlation. the green module showed positive correlation with ad for sodium ion transport. ad has been reported to be associated with impaired glutamate clearance and depressed na+/k+ atpase levels in the brain. several solute carrier family members were found in the green module. the pink module showed correlations with ad and disease stage. genes related to ion homeostasis and immune responses were identified in the pink module. metal ion dyshomeostasis is a well-recognized cofactor in several neurodegenerative disorders. in ad, the process of aβ misfolding and plaque aggregation is greatly influenced by alterations in the homeostasis of the aforementioned metal ions. a strong genetic association between aβ formation and ad has been reported in several studies and we found that aβ is highly expressed in the ad mouse model used in this study. the yellow module was correlated with ad and included genes related to cell death, apoptosis, and development. ad is correlated with region-specific neuronal cell loss. notch found in the yellow module, is known to regulate the notch signaling pathway, which is involved in controlling the fate of neural and non-neural cells in development process and adults. several observations suggest that enhanced notch signaling and expression could be instrumental in neurodegeneration in ad. huntingtin interacting protein, found in the yellow module, is related to cell death through caspase- activation in immortalized hippocampal neuroprogenitor cells. thus, the functions of the proteins in the green, pink, and yellow modules are directly associated with ad. the black, brown and red modules showed relationships with disease stage and are involved in brain development including central nervous system development, neurogenesis, and forebrain development. the blue and turquoise modules showed significant relationships with ad and disease stage. the gene list for each of the modules and the gsa results are presented in additional file 1: table s and additional file 2: table s mirna-mrna regulatory network in tg mice we constructed a co-expression network from the functional gene module to study mirna-mrna interactions and the regulation of gene expression by mirna. we integrated several approaches to predict mirna targets and module regulators, which were predicted using 3-target site analysis. although black, brown, and yellow modules did not show direct correlations with genetic ad, these modules were considered as ad related mirna modules because dysregulation of neurogenesis often cause neurodegenerative diseases. using all these modules, we calculated all the pair expression correlations from target relations using sequence-based target prediction results and selected mirna-mrna interactions by anti-correlation. the constructed co-expression weighted network contains the black, brown, red, and pink modules. mrnas in the red module correlate significantly with disease stage. mir-34b, which interacts with genes in the brown and pink modules, has been proposed as a key regulator to integrate age-associated diseases in the vertebrate brain. it closely interacted with mrnas in the brown and pink modules. some genes in the disease stage-related modules have been reported to be associated with ad. for example, the members of the black and red modules are related to neurogenesis in normal developmental stages. the ad network was constructed using the yellow and green modules. in particular, mir- and mir- are known to be down-regulated in ad patients.figure the subnetwork of weighted coexpression networks of mirna-mrna in ad. the subnetwork of brown, grey, and pink module genes and related mirnas. as presented in figure  transgenic plots, these modules show negative correlations. the subnetwork of green and yellow module genes and related mirnas. the subnetwork of weighted coexpression networks. a compact network was constructed from arrestin domain containing neighbors and related mirnas to their target genes. the interaction between mirna and their target genes were predicted by sequence analysis and expression anti-correlation. significant interactions of mirna-mrna were predicted by the co-expression network. we assigned priority to mirna-mrna targets based on network topology. we assumed that highly ranked genes in mirna-mrna networks are more likely to play critical roles in the pathophysiology of ad. using the pagerank algorithm, which is useful for describing the relative node importance in a network connection, we measured the pagerank score for mirna-mrna interactions and assigned the rank of interaction priority using only differentially expressed mirnas and deg. we determined that mirna-mrna interactions were significant targets and confirmed mirnas with a high pagerank score of <. microglia induction in ad can be explained by our mirna-mrna interaction prediction system. our results predicted that mir-34b was involved in the regulation of colony stimulating factor receptor, which has been reported to be critical for survival and proliferation of microglia. our microarray data showed that csf1r was significantly down-regulated. aging and neurodegeneration seem to be modulated by mir- expression through microglial activation and activation of microglial chemokine receptor is known to lead to neuronal death in ad. our current microarray data demonstrate that expression of chemokine ligand and adam metallopeptidase domain increased in tg mice at 8 months of age. adam is a predicted target of mir- and mir- and is involved in cx3cl cleavage. cleavage of cx3cl by adam can lead to microglial activation of neuronal degeneration. differential expression of mirna in tg mice in total, differentially expressed mirnas were identified in the early- and late-stage ad mouse model. we identified several mirnas that correlated with ad. for example, mir- which is down-regulated in the brains of ad mice, is specifically expressed in the brain and has been demonstrated to negatively regulate dendritic spine formation in vitro. mir- is enriched at synapses and modulate synaptic development and spine size through the control of acyl-protein thioesterase- and the depalmitoylation of g protein alpha, a down-stream target of apt which is an activator of rho down-stream of g-protein coupled receptor. mir-146b is down-regulated in ad mice and has been reported to be consistently altered in both hippocampus and medial frontal gyrus in ad mouse models. mir-146b could be inferred as a negative regulator of innate immunity and its down-regulation in the ad brain provides support for an induction of toll like receptor signaling in ad. mir- is highly expressed in the cortex during early fetal development and is related to the growth spurt in the cerebellum compared to the cortex. mir-26a was observed to be down-regulated in tg mice at months of age. mir-26a has been reported to be down-regulated in nasopharyngeal carcinomas and to suppress cell proliferation and colony formation by inducing g1-phase cell-cycle arrest. mir-34b and mir-34c were up-regulated in our ad model mice and have been reported to be up-regulated in ad patients. we validated the expression levels of three mirnas from the list of candidates. real time-pcr results showed that at months of age, mir139-5p expression showed tendency to increase, in the hippocampi of tg mice compared to littermate controls. the expression of mir340-5p and mir3470a did not increase significantly in mutant tg mice at either or months of age.figure verification of the expression of mrna and mirna in the hippocampus of tg mice. the mrna levels of arrdc protein phosphatase regulatory subunit 3c and splicing factor, proline- and glutamine-rich in the hippocampus of tg mice were verified using rt-pcr. or mutant tg mice; §: p <  between lm or mt mice; §§: p <  between lm or mt mice). the expression levels of mir139-5p, mir340-5p, and mir3470a in the hippocampus of lm or mt mice were determined using real-time pcr. differential expression of mrna in tg mice: arrdc ppp1r3c, sfpq and metabolic dysfunction using integration analysis, we predicted functions of mrna and mirna associations in ad pathology. focusing on that many neurodegenerative diseases are known to be caused by metabolic dysfunction, we decided to validate our prediction system through bio-energy metabolism related genes such as arrestin domain containing, a predicted target of mir- mir- mir- and mir-3470a, in the brown module. to validate the network topology analysis system, we selected significant interactions in non-demir and determined the target gene expression. the mrna level of arrdc was increased in the hippocampus of tg mice at 4 months of age compared to their littermate controls. the mrna level of arrdc was significantly increased in an age-dependent manner in the hippocampus of lm controls. arrdc mrna also showed an age-dependent increase in mt mice. interestingly, we determined that protein phosphatase regulatory subunit 3c exhibited direct relationships with age or ad mutation in consequence map data. the mrna levels of ppp1r3c increased with age in lm controls and had a tendency to increase in mt mice at 4 months of age compared to lm controls. we determined that mrna levels of ppp1r3c and splicing factor proline and glutamate-rich decreased with age in mt mice and also decreased in mt mice at the late symptomatic stage compared to lm controls. we next determined arrdc and sfpq protein expression levels by performing semi-quantitative western blot analysis using the hippocampus samples from mt mice and lm controls at 10 months of age. the protein expression levels of arrdc and sfpq decreased in mt mice compared to lm controls. co-immunoprecipitation data showed direct molecular interaction between arrdc and sfpq proteins and this interaction was increased in mt mice compared to lm controls.figure molecular interaction between arrdc and sfpq proteins. the protein levels of arrdc and sfpq in the hippocampus of tg mice were determined using western blot. the molecular interaction between arrdc and sfpq proteins was detected by co-immunoprecipitation experiment. arrdc has been implicated in the regulation of body mass and energy metabolism and plays a role as a tumor suppressor. as a down-stream target of arrdc sfpq has been implicated in cellular processes such as transcriptional regulation and rna splicing, both of which are related to circadian rhythms. sfpq is expressed in neurons and glia and is confined to the nuclei in both cell types. ppp1r3c could be induced by hypoxia as a hypoxia induced factor -targeted gene. protein phosphatase is known to target glycogen and to regulate glycogen metabolism. arrdc ppp1r3c, and sfpq belong to a metabolism-related category of genes, as we demonstrated in the brown module. previously, poll et al. demonstrated that arrestin-dependent gpcr signaling is controlled by rapid dephosphorylation of pp. for functional validation of arrdc we overexpressed arrdc by transfection or silenced arrdc expression with sirna in primary cultured cortical neurons from tg mice and littermate controls. arrdc overexpression increased the mrna levels of arrdc ppp1r3c, and sfpq in the cortical neurons of both lm and mt mice. after silencing with arrdc sirna, the mrna levels of ppp1r3c and sfpq decreased in mt cortical neurons. real time-pcr results showed that mir139-5p and mir3470a expressions have tendency to decrease after arrdc overexpression in both lm and mt primary cortical neurons. we also determined the length and the number of neurites in primary cultured neurons from tg mice using anti-tau antibody. the number of tau-positive neurons from mt mice significantly decreased and the length of neurites decreased as well compared to lm primary neurons. however, arrdc overexpression could not alter the length or the number of neurites compared to vehicle treated mt neurons. to determine arrdc sfpq, and ppp1r3c mrna levels and mirna interaction in aβ toxic condition, we administered 2 μm aβ into the primary cultured cortical neurons from tg mice. after aβ treatment, the mrna levels of arrdc ppp1r3c, and sfpq increased in the cortical neurons of both lm and mt mice. real time-pcr results showed that after aβ treatment, mir139-5p and mir3470a expressions have tendency to increase in cortical neurons of tg mice compared to vehicle treated mt neurons. to detect luciferase activity of arrdc promoter in toxic condition, we transfected arrdc promoter into primary cultured cortical neurons of tg mice then administered aβ. luciferase activity for arrdc promoter significantly increased in mt cortical neurons compared to lm control. also after aβ treatment, luciferase activity for arrdc promoter significantly increased in lm cortical neurons compared to vehicle treatment.figure validation of arrdc function in primary cultured cortical neurons from tg transgenic mice. overexpression of arrdc gene in primary cultured cortical neurons from tg mice. rt-pcr results show that the mrna levels of ppp1r3c and sfpq were increased by arrdc overexpression. knock-down of arrdc gene in primary cultured cortical neurons from tg mice. the mrna levels of ppp1r3c and sfpq were decreased by the treatment of arrdc sirna. real-time pcr data show that the expression levels of mir139-5p and mir3470a had decreasing tendency in the primary cortical neurons of tg mice. immunocytochemical staining show no significant enhancement in the number and length of neurites in arrdc overexpressed primary cortical neurons from tg mice. determination of the expression of arrdc related mrna/mirna under a administration. rt-pcr show that the mrna levels of arrdc ppp1r3c and sfpq were increased by aβ administration in primary cultured cortical neurons of tg mice. real time pcr show that the expression levels of mir139-5p and mir3470a were not significantly altered by aβ administration in primary cortical neurons from tg mice. luciferase activity of arrdc promoter increased after aβ treatment in lm cortical neurons but not in mt neurons. taken together, these data suggest that arrdc ppp1r3c, and sfpq interact with each other in regulating the age- and disease-specific gene expression of similar biological pathways such as bioenergetics and metabolism, which are very strong candidate categories implicated in the pathophysiological progressive changes of neurodegeneration.  our results demonstrate ad-specific changes in the mirna regulatory system and a relationship between the levels of mirnas and their targets in the ad hippocampus. we identified ad-related modules and provided a specific network that integrates differential mirna and mrna expression profiles. we proposed relevant mirna-mrna target relationships in an ad based on network topological priorities and mirna mediated mechanism in ad pathology. our data highlight the functions and mechanisms of various mirnas and their target genes in the molecular pathology of ad including the genes which are related in ion transport, ion homeostasis, immune responses, cell death, apoptosis, development, and bio-energy metabolism. knowledge of candidate genes identified through the simultaneous genome-wide profiling of mirna and mrna may be useful for the development of future therapeutics in ad. 
these data help further our understanding of the function and mechanism of various mirnas and their target genes in the molecular pathology of ad. our results demonstrate ad-specific changes in the mirna regulatory system as well as the relationship between the expression levels of mirnas and their targets in the hippocampus of tg mice. for functional validation in primary cultured neurons from tg mice and littermate controls, the overexpression of arrdc enhanced ppp1r3c expression. pathological environment created by aβ treatment increased the gene expression of ppp1r3c and sfpq but did not significantly alter the expression of mir139-5p or mir3470a. we constructed gene regulatory networks and used the pagerank algorithm to predict significant interactions between mirna and mrna. among these, significant mirna-mrna interactions were predicted by integrating sequence target prediction, expression analysis, and the pagerank algorithm. here, we applied the joint profiling analysis of mrna and mirna expression levels to tg ad model mice at and 8 months of age using a network topology-based method. we identified a set of mirna-mrna interactions that were changed in the hippocampus of tg ad model mice. aβ treatment increased the promoter activity of arrdc gene in lm primary cells but not in mt primary cells. we determined the expression levels of several candidate genes and mirna. sequence-based target prediction algorithms and anti-correlation profiles have been applied to predict mirna targets using omics data, but this approach often leads to false positive predictions.
 little is known about the relationship between mirna and mrna expression in alzheimer’s disease at early- or late-symptomatic stages. sequence-based target prediction algorithms and anti-correlation profiles have been applied to predict mirna targets using omics data, but this approach often leads to false positive predictions. here, we applied the joint profiling analysis of mrna and mirna expression levels to tg ad model mice at and 8 months of age using a network topology-based method. we constructed gene regulatory networks and used the pagerank algorithm to predict significant interactions between mirna and mrna.  in total, cluster modules were predicted by the transcriptome data for co-expression networks of ad pathology. in total, mirnas were identified as being differentially expressed in ad. among these, significant mirna-mrna interactions were predicted by integrating sequence target prediction, expression analysis, and the pagerank algorithm. we identified a set of mirna-mrna interactions that were changed in the hippocampus of tg ad model mice. we determined the expression levels of several candidate genes and mirna. for functional validation in primary cultured neurons from tg mice and littermate controls, the overexpression of arrdc enhanced ppp1r3c expression. arrdc overexpression showed the tendency to decrease the expression of mir139-5p and mir3470a in both lm and mt primary cells. pathological environment created by aβ treatment increased the gene expression of ppp1r3c and sfpq but did not significantly alter the expression of mir139-5p or mir3470a. aβ treatment increased the promoter activity of arrdc gene in lm primary cells but not in mt primary cells.  our results demonstrate ad-specific changes in the mirna regulatory system as well as the relationship between the expression levels of mirnas and their targets in the hippocampus of tg mice. these data help further our understanding of the function and mechanism of various mirnas and their target genes in the molecular pathology of ad. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords alzheimer’s diseasemicrornatranscriptomenetwork moduleissue-copyright-statement© the author 2014 