BACKGROUND
amyloid precursor protein  is the parent molecule of the neurotoxic amyloid-β, implicated in the aetiology of alzheimer’s disease.
amyloid-β is generated by sequential cleavage of app by β and γ-secretases.
however, the likely predominant route of app processing is via α-secretase  <cit> , which not only precludes amyloid-β production but also generates secreted amyloid precursor protein alpha   <cit> .
this molecule interacts with the β-secretase, bace <dig>  and directly inhibits amyloid-β production  <cit> .
furthermore, mounting evidence implicates sappα in a wide variety of neuronal processes.
it has been shown to both protect against glutamate toxicity in rat hippocampal neurons in vitro and in rat models of traumatic brain injury in vivo <cit>  and promote neurite outgrowth in vitro <cit> .
furthermore, sappα has recently been shown to not only direct human embryonic stem cells into neuronal precursor cells  <cit> , but to increase proliferation of neural precursor cells from the rat hippocampus in vitro, and promote their proliferation in the mouse hippocampus in vivo <cit> .
additionally, sappα has been shown to enhance synaptic plasticity and restore memory deficits in rats and mice in vivo <cit> .
while a definitive cognate receptor for sappα has not yet been identified, sappα is known to activate intracellular signalling cascades in both neurons and glia and enhance synaptic protein synthesis in vitro <cit> .
as the reported physiological effects of sappα, are dependent on rapid and persistent alterations in gene expression, it is also likely that regulation of transcription is integral to the function of sappα.
recently, expression of the transcription factor egr <dig> was shown to mediate sappα stimulated axonal outgrowth of primary neurons from mice in vitro <cit> , and sappα has previously been shown to induce the expression of several neuroprotective genes in mouse organotypic hippocampal slices  <cit> .
this observation is particularly important as the hippocampus is a region vulnerable to the early neurodegenerative changes observed in alzheimer’s disease.
interestingly, however, sappα knock-in mice show significantly lower expression of the plasticity related genes arc, egr <dig> and fos.
furthermore, in general, the gene expression profiles of app knockout and sappα knock-in mice do not differ substantially  <cit> , questioning the role of the n-terminal region of app in regulation of gene expression.
in light of these somewhat conflicting data we sought to determine whether the application of sappα to organotypic hippocampal slice cultures for up to  <dig> h alters gene expression, and if so, whether the gene networks or biological pathways identified reflected the known physiological roles of sappα.
we found that sappα rapidly enhances the transcription environment and alters subsequent gene expression in a manner that is likely to underpin the reported neuroprotective effects of sappα.
RESULTS
sappα protects against n-methyl d-aspartate toxicity
we have previously shown that purified human recombinant sappα produced in our laboratory  <cit>  regulates protein synthesis  <cit> , synaptic plasticity and memory  <cit> , as well as neurogenesis  <cit> .
however, prior to determining the effects of sappα on gene expression, we sought to extend our assessment of the functionality of recombinant sappα.
accordingly, we tested whether sappα could protect against n-methyl-d-aspartate -induced excitotoxicity in hippocampal organotypic slice cultures.
hippocampal organotypic slice cultures  were challenged with nmda  and cell death was assessed by measurement of propidium iodide  fluorescence  <dig> h later.
in phosphate buffered saline -treated control cultures, the tissue had low pi fluorescence across all hippocampal regions, indicating little cell death .
in contrast, nmda treatment caused significantly higher levels of pi fluorescence in cornu ammonis area  <dig> and  <dig>  and the inner and outer blades of the dentate gyrus  relative to the pbs-treated control cultures , indicating induction of extensive cell death.
slices were incubated with  <dig> ,  <dig> ,  <dig>   <dig> nm sappα or pbs-control for  <dig> h prior to, and for  <dig> h following nmda insult.
incubation with  <dig> nm sappα resulted in a significant reduction in pi fluorescence in ca <dig>  and the inner  and outer blades  of the dg compared with nmda treatment alone , indicating a neuroprotective effect.
no significant effects were detected following incubation with the other sappα + nmda concentrations but sappα exposure trended towards a u-shaped dose–response curve .
together, these findings indicate that  <dig> nm sappα increases cell survival following nmda exposure.
although sappα has previously been shown to protect against glutamate toxicity  <cit> , glutamate and nmda may induce excitotoxicity by non-overlapping signaling pathways  <cit> , and so this study extends the known neuroprotective effects of sappα and validates the activity of sappα in our model system.
regulation of gene expression by sappα
to investigate the temporal effects of sappα on the hippocampal transcriptome, hippocampal organotypic slice cultures were treated with  <dig> nm sappα  or pbs for either  <dig> min,  <dig> h or  <dig> h and expression profiling was performed with affymetrix rat gene  <dig>  st arrays .
data analysis, using dual selection criteria  and fold change ±  <dig> ), identified temporally distinct sappα-induced patterns of expression , a subset of which was confirmed by quantitative pcr .
the majority of genes were modestly changed , an effect particular to neurobiological tissue  <cit>  and consistent with previous studies  <cit> .
interestingly, two thirds  of differentially expressed genes were upregulated in the  <dig> min dataset, while the majority of differentially expressed genes were downregulated at  <dig> h  and  <dig> h  , suggesting that there is a relatively rapid onset homeostatic control of gene expression.
overall, these data confirm that sappα, the n-terminal region of app, regulates gene expression in a time-dependent manner.
to explore further how the effects of sappα on gene expression changed with time, the expression profiles were examined using a venn diagram.
this analysis showed that the three datasets were largely distinct .
only two genes were in common across all three datasets and only one was annotated.
interestingly, this gene, acyl-protein thioesterase  <dig>   showed a distinct temporal response to sappα treatment, decreasing in expression at  <dig> min , increasing at  <dig> h  and decreased by  <dig> h .
activation of apt <dig> results in depalmitoylation of substrate proteins, a process important in regulating subcellular localisation of plasticity related ion channels and gap <dig>  <cit> .
thus, these data predict that regulation of apt <dig> contributes to the plasticity-related effects of sappα.
as sappα enhances synaptic plasticity and restores memory deficits  <cit> , we next investigated whether there was a significant crossover between our sappα-regulated datasets and those regulated by synaptic plasticity.
previously, we identified distinct patterns of gene expression elicited by hippocampal long-term potentiation , a widely accepted model of memory mechanisms and synaptic plasticity  <cit> .
here, we show that there was little intersection between the genes within the datasets .
for example, there were only five rapidly responding genes in common across the early time-points analysed  and the fold change in these overlapping genes was relatively curtailed in the sappα dataset .
despite this, when we compared biological functions and pathways between the two data sets, we found that they contained overlapping functions related to gene expression , development  and neurogenesis .
this suggests that while there was little overlap in the actual genes regulated, there is overlap in some of the biological functions controlled by sappα and ltp.
as we found that sappα-induced a rapid upregulation followed by a generalised downregulation of gene expression, we hypothesised that this temporal pattern may be regulated by microrna.
a subset of probes on the affymetrix rat gene  <dig>  st arrays represent stem loop microrna sequences and therefore allow analysis of primary and precursor microrna.
we found evidence for regulation of  <dig> microrna in response to sappα , the majority of which were upregulated at  <dig> min, suggesting that the mature microrna may contribute to the later observed downregulation of gene expression.
in accordance with this, we found no evidence for regulation by microrna within the  <dig> min dataset using the web application mirvestigator framework  <cit> .
however, analysis of the  <dig> h dataset identified mir- <dig>  as a candidate microrna controlling the expression of a subset of mrna .
as the mir- <dig> primary transcript was increased at  <dig> min  this suggests a role for mir- <dig> in sappα-mediated gene expression, via mrna degradation.
furthermore, mirvestigator predicted regulation by mir-201*  within the  <dig> h dataset , although we found no evidence for altered levels of the primary microrna transcript in our analyses.
as production of the alzheimer’s disease-related amyloid-ß and sappα are mutually exclusive, we analysed how alzheimer’s disease-associated genes were affected in our three data sets.
to achieve this we compared our datasets with the alzheimer gene database, alzgene  <cit>  and carried out an in-depth literature search.
we identified a group of mrna and microrna previously associated with amyloid-ß induced toxicity , or implicated in alzheimer’s disease processes,   <cit> , .
as the expression of these genes was opposite to that observed in alzheimer’s disease this analysis suggests that sappα may act, in part, through the same pathways as amyloid-ß.
sappα rapidly regulates transcriptional processes
to systematically investigate the functional significance of the genes rapidly regulated by sappα, we used the ingenuity pathway analysis  software to identify gene networks from the  <dig> min dataset.
gene expression was identified as the most significant biological function of the highest scoring network, with the majority of the genes in this network upregulated .
analysis of this network showed multiple mechanisms engaged by sappα to upregulate gene expression.
this includes inducible transcription factors of the ap- <dig> complex  <cit>   and the early growth response transcription factor, egr <dig>  enhanced expression of fos and egr <dig> was confirmed by qpcr ; furthermore, the upstream regulatory analysis tool within ipa predicted downregulation of a negative regulator of ap- <dig> activity fosl1/fra <dig>   <cit> , an effect likely to further amplify the sappα-induced transcriptional response .
this network also predicts that sappα mediates transcription via activation of the plasticity-related transcription factors, camp response element binding protein  and nuclear factor kappa b , that form central hubs within the network.
while their expression levels remain unchanged, this is highly consistent with their roles as constitutive transcription factors regulated by post-transcriptional modification.
indeed, this network includes dusp <dig> , a plasticity-related phosphatase and map3k <dig> , a kinase known to phosphorylate and activate nf-κb, as well as the nf-κb regulatory molecule, nf-κbid   <cit> .
together these data suggest that sappα treatment not only enhances the levels of transcription factors but also contributes to a dynamic interplay between a subset of protein kinases and phosphatases likely to maintain transcriptional activity.
regulation of the chromatin environment is a third predicted mechanism by which sappα regulates expression of genes in this network.
the network contains mediators of chromatin condensation  acetyltransferase; + <dig>  fold) and histone biotinylation ) ligase; + <dig>  fold)  <cit> .
upregulation of kat2b is likely to result in reduced condensation of chromatin, occurring via acetylation of tnp <dig>  a molecule also regulated within this network.
to further explore the  <dig> min dataset we used the functional annotation tool, david.
this highlighted cognition and sensory perception  as well as response to corticosterone  as significantly enriched functions .
interestingly, there is an increase in basal glucocorticoid levels in alzheimer’s disease,  <cit> .
thus sappα may regulate the expression of genes associated with this pathway.
sappα-induced regulation of apoptosis and the inflammatory response
we next explored the gene networks affected by exposure to sappα for extended times.
following  <dig> h exposure to sappα, inflammatory response was the most significant biological function of the highest scoring network formed used ipa .
this network contains the proinflammatory cytokine il1a, previously associated with alzheimer’s disease, development and plasticity  <cit> , immune associated genes  and chemokines .
these chemokines are involved in chemoattraction of immune cells to sites of tissue damage but have widespread non-immunological effects in the central nervous system, including regulation of neural cell proliferation, migration, survival and synaptic transmission  <cit> .
that sappα treatment promotes regulation of chemokine responses is also supported from analysis of the  <dig> h data set with the functional annotation tool, david .
like the  <dig> min network, nf-κb forms a central hub in this network, however, many of the genes contributing to the hub, including ehf , which promotes apoptosis  <cit> , are downregulated.
interestingly, using the upstream regulatory element analysis tool to analyse the genes upregulated within the  <dig> h dataset, it is predicted that sappα induces inhibition of the transcription factor, tumour protein  <dig> , which is also an important regulator of apoptosis, and is increased in alzheimer’s disease  <cit>  .
in addition, upstream regulatory element analysis using the entire  <dig> h dataset, predicts activation of the transcription factors ap- <dig>  hmgb <dig>  and stat <dig>   suggesting other mechanisms by which sappα coordinates this gene response.
sappα-induced long-term regulation of neurogenic, inflammatory response and cell survival pathways
as the reported biological functions of sappα, such as neuroprotection and regulation of memory, are enduring physiological changes, we explored the gene expression response induced by a  <dig> h exposure to sappα.
to identify the functional relationships within the  <dig> h differentially expressed gene list, we used the david functional annotation tool.
this analysis identified neurogenesis, morphogenesis and development as important biological functions affected  <dig> h post-sappα treatment  .
we extended analysis of this dataset by interpretation of the highest scoring network derived using ipa, .
this network was composed of genes with overlapping biological functions, including upregulated genes related to cell proliferation , regulation of genes likely to promote cell survival  and inhibit apoptosis .
as sappα has previously been shown to promote growth, survival and proliferation in neurons and stem cells  <cit>  these findings supports a role for sappα-induced gene expression in mediating these events.
discussion
in order to better understand the role that regulation of gene expression may play in mediating the wide-ranging cellular effects of sappα, we performed a global transcriptome analysis in organotypic hippocampal slice cultures treated with  <dig> nm sappα for  <dig> min,  <dig> h or  <dig> h. we focused on the hippocampus as this region is essential to learning and memory and is especially vulnerable to degeneration in alzheimer’s disease  <cit> .
our analysis demonstrated that sappα rapidly regulates gene expression by engaging multiple transcriptional regulatory mechanisms.
this is evidenced by rapid and transient upregulation of inducible transcription factors , temporally specific regulation of constitutively expressed transcription factors  and microrna, as well as, modulation of the chromatin environment.
network analysis showed that regulation of nf-κb is a consistent theme across all three time-points investigated.
this supports previous work proposing that the neuroprotective effects of sappα are mediated through activation of nf-κb and subsequent enhanced activation of gene expression  <cit> .
sappα-induced nf-κb activation is implicated in protecting neural cells from apoptosis, therefore our results suggest a role for specifically regulated genes in this process  <cit> .
interestingly, nf-κb has recently been intimately linked with amyloid-ß production  <cit>  and sappα modulates app processing  <cit> .
therefore, the control of amyloid-β production may be crucially dependent on the activation status of nf-κb, over which sappα has influence  <cit> .
increasing evidence points to the involvement of dysregulated microrna in alzheimer’s disease  <cit>  and indeed an nf-κb-sensitive microrna has been implicated in modulating the inflammatory circuit in alzheimer’s disease  <cit> .
our results highlight a role for microrna in sappα-induced regulation of gene expression.
we found that a short-term exposure to sappα was associated with upregulation of a subset of microrna.
as medium to long-term sappα exposure resulted in a general decrease in gene expression, we propose that this may be mediated by microrna.
interestingly, we reported a similar general effect on gene expression following the induction of ltp, which we have shown to be associated with altered levels of mature microrna  <cit> .
our results also demonstrate that sappα induces a subset of plasticity-associated immediate early genes, however, we found little evidence that the sappα-induced gene response paralleled that of the ltp gene response.
therefore, post-translational modifications, and altered synaptic protein synthesis, are likely to mediate many of the effects of sappα on ltp  <cit> .
it is of particular note that prolonged exposure to sappα resulted in persistent effects on gene expression that correlate closely with the documented neuroprotective and neurogenic roles of sappα.
this interpretation is consistent with the only other microarray analysis of sappα-regulated gene expression in the hippocampus  <cit> .
this study reported that treatment of mouse organotypic slices with  <dig> nm sappα for  <dig> h also resulted in regulation of several neuroprotective genes.
interestingly, there was little coherence between the genes identified in this study and our own.
this may result from differences in the array type used , the number of annotated genes investigated , the statistical analyses performed  or species .
while there is a paucity of data estimating the physiological levels of sappα, it has been estimated to fall within the pm range within plasma and brain homogenates  <cit> , however, it is difficult to extrapolate these concentrations to the ex vivo model used in this study.
our studies highlight that  <dig> nm sappα is sufficient to modulate gene expression in ex vivo models and emphasizes that both medium and longer-term exposure to sappα elicits an inflammatory and immune gene response, which likely provides a neuroprotective environment.
this neuroprotective setting appears to be strengthened by a parallel downregulation of apoptotic pathways and increases in cell proliferation and survival.
furthermore, we found evidence for the regulation of genes associated with neurogenesis.
recent evidence has also linked sappα with the induction of neurogenesis in the mammalian brain  <cit> .
as new brain cells have the capacity to integrate into previously established neural networks and contribute to hippocampal functioning  <cit> , neurogenesis may also contribute to the sappα-induced neuroprotective and memory enhancing effects over the long-term.
CONCLUSIONS
in summary, our analyses consolidate the concept that sappα regulates gene expression.
we provide evidence that this occurs in a temporally specific manner, and occurs through providing an environment conducive for transcription that results in activation of immediate early gene transcription factors, known to mediate neuroprotection and proliferation  <cit> , and regulation of microrna.
furthermore, we demonstrate that gene networks constructed following medium and prolonged exposure to sappα reveal novel mechanisms likely to underpin and consolidate the neuroprotective stimulus induced by sappα in the hippocampus.

 amyloid precursor protein is the parent molecule of the neurotoxic amyloid-β, implicated in the aetiology of alzheimer’s disease. amyloid-β is generated by sequential cleavage of app by β and γ-secretases. however, the likely predominant route of app processing is via α-secretase, which not only precludes amyloid-β production but also generates secreted amyloid precursor protein alpha. this molecule interacts with the β-secretase, bace and directly inhibits amyloid-β production. furthermore, mounting evidence implicates sappα in a wide variety of neuronal processes. it has been shown to both protect against glutamate toxicity in rat hippocampal neurons in vitro and in rat models of traumatic brain injury in vivo and promote neurite outgrowth in vitro. furthermore, sappα has recently been shown to not only direct human embryonic stem cells into neuronal precursor cells, but to increase proliferation of neural precursor cells from the rat hippocampus in vitro, and promote their proliferation in the mouse hippocampus in vivo. additionally, sappα has been shown to enhance synaptic plasticity and restore memory deficits in rats and mice in vivo. while a definitive cognate receptor for sappα has not yet been identified, sappα is known to activate intracellular signalling cascades in both neurons and glia and enhance synaptic protein synthesis in vitro. as the reported physiological effects of sappα, are dependent on rapid and persistent alterations in gene expression, it is also likely that regulation of transcription is integral to the function of sappα. recently, expression of the transcription factor egr was shown to mediate sappα stimulated axonal outgrowth of primary neurons from mice in vitro, and sappα has previously been shown to induce the expression of several neuroprotective genes in mouse organotypic hippocampal slices. this observation is particularly important as the hippocampus is a region vulnerable to the early neurodegenerative changes observed in alzheimer’s disease. interestingly, however, sappα knock-in mice show significantly lower expression of the plasticity related genes arc, egr and fos. furthermore, in general, the gene expression profiles of app knockout and sappα knock-in mice do not differ substantially, questioning the role of the n-terminal region of app in regulation of gene expression. in light of these somewhat conflicting data we sought to determine whether the application of sappα to organotypic hippocampal slice cultures for up to h alters gene expression, and if so, whether the gene networks or biological pathways identified reflected the known physiological roles of sappα. we found that sappα rapidly enhances the transcription environment and alters subsequent gene expression in a manner that is likely to underpin the reported neuroprotective effects of sappα.  sappα protects against n-methyl d-aspartate toxicity we have previously shown that purified human recombinant sappα produced in our laboratory regulates protein synthesis, synaptic plasticity and memory, as well as neurogenesis. however, prior to determining the effects of sappα on gene expression, we sought to extend our assessment of the functionality of recombinant sappα. accordingly, we tested whether sappα could protect against n-methyl-d-aspartate -induced excitotoxicity in hippocampal organotypic slice cultures. hippocampal organotypic slice cultures were challenged with nmda and cell death was assessed by measurement of propidium iodide fluorescence h later. in phosphate buffered saline -treated control cultures, the tissue had low pi fluorescence across all hippocampal regions, indicating little cell death. in contrast, nmda treatment caused significantly higher levels of pi fluorescence in cornu ammonis area and and the inner and outer blades of the dentate gyrus relative to the pbs-treated control cultures, indicating induction of extensive cell death. slices were incubated with,, nm sappα or pbs-control for h prior to, and for h following nmda insult. incubation with nm sappα resulted in a significant reduction in pi fluorescence in ca and the inner and outer blades of the dg compared with nmda treatment alone, indicating a neuroprotective effect. no significant effects were detected following incubation with the other sappα + nmda concentrations but sappα exposure trended towards a u-shaped dose–response curve. together, these findings indicate that nm sappα increases cell survival following nmda exposure. although sappα has previously been shown to protect against glutamate toxicity, glutamate and nmda may induce excitotoxicity by non-overlapping signaling pathways, and so this study extends the known neuroprotective effects of sappα and validates the activity of sappα in our model system. regulation of gene expression by sappα to investigate the temporal effects of sappα on the hippocampal transcriptome, hippocampal organotypic slice cultures were treated with nm sappα or pbs for either min, h or h and expression profiling was performed with affymetrix rat gene st arrays. data analysis, using dual selection criteria and fold change ±  ), identified temporally distinct sappα-induced patterns of expression, a subset of which was confirmed by quantitative pcr. the majority of genes were modestly changed, an effect particular to neurobiological tissue and consistent with previous studies. interestingly, two thirds of differentially expressed genes were upregulated in the min dataset, while the majority of differentially expressed genes were downregulated at h and h, suggesting that there is a relatively rapid onset homeostatic control of gene expression. overall, these data confirm that sappα, the n-terminal region of app, regulates gene expression in a time-dependent manner. to explore further how the effects of sappα on gene expression changed with time, the expression profiles were examined using a venn diagram. this analysis showed that the three datasets were largely distinct. only two genes were in common across all three datasets and only one was annotated. interestingly, this gene, acyl-protein thioesterase showed a distinct temporal response to sappα treatment, decreasing in expression at min, increasing at h and decreased by h. activation of apt results in depalmitoylation of substrate proteins, a process important in regulating subcellular localisation of plasticity related ion channels and gap. thus, these data predict that regulation of apt contributes to the plasticity-related effects of sappα. as sappα enhances synaptic plasticity and restores memory deficits, we next investigated whether there was a significant crossover between our sappα-regulated datasets and those regulated by synaptic plasticity. previously, we identified distinct patterns of gene expression elicited by hippocampal long-term potentiation, a widely accepted model of memory mechanisms and synaptic plasticity. here, we show that there was little intersection between the genes within the datasets. for example, there were only five rapidly responding genes in common across the early time-points analysed and the fold change in these overlapping genes was relatively curtailed in the sappα dataset. despite this, when we compared biological functions and pathways between the two data sets, we found that they contained overlapping functions related to gene expression, development and neurogenesis. this suggests that while there was little overlap in the actual genes regulated, there is overlap in some of the biological functions controlled by sappα and ltp. as we found that sappα-induced a rapid upregulation followed by a generalised downregulation of gene expression, we hypothesised that this temporal pattern may be regulated by microrna. a subset of probes on the affymetrix rat gene st arrays represent stem loop microrna sequences and therefore allow analysis of primary and precursor microrna. we found evidence for regulation of microrna in response to sappα, the majority of which were upregulated at min, suggesting that the mature microrna may contribute to the later observed downregulation of gene expression. in accordance with this, we found no evidence for regulation by microrna within the min dataset using the web application mirvestigator framework. however, analysis of the h dataset identified mir- as a candidate microrna controlling the expression of a subset of mrna. as the mir- primary transcript was increased at min this suggests a role for mir- in sappα-mediated gene expression, via mrna degradation. furthermore, mirvestigator predicted regulation by mir-201* within the h dataset, although we found no evidence for altered levels of the primary microrna transcript in our analyses. as production of the alzheimer’s disease-related amyloid-ß and sappα are mutually exclusive, we analysed how alzheimer’s disease-associated genes were affected in our three data sets. to achieve this we compared our datasets with the alzheimer gene database, alzgene and carried out an in-depth literature search. we identified a group of mrna and microrna previously associated with amyloid-ß induced toxicity, or implicated in alzheimer’s disease processes,,. as the expression of these genes was opposite to that observed in alzheimer’s disease this analysis suggests that sappα may act, in part, through the same pathways as amyloid-ß. sappα rapidly regulates transcriptional processes to systematically investigate the functional significance of the genes rapidly regulated by sappα, we used the ingenuity pathway analysis software to identify gene networks from the min dataset. gene expression was identified as the most significant biological function of the highest scoring network, with the majority of the genes in this network upregulated. analysis of this network showed multiple mechanisms engaged by sappα to upregulate gene expression. this includes inducible transcription factors of the ap- complex and the early growth response transcription factor, egr enhanced expression of fos and egr was confirmed by qpcr; furthermore, the upstream regulatory analysis tool within ipa predicted downregulation of a negative regulator of ap- activity fosl1/fra, an effect likely to further amplify the sappα-induced transcriptional response. this network also predicts that sappα mediates transcription via activation of the plasticity-related transcription factors, camp response element binding protein and nuclear factor kappa b, that form central hubs within the network. while their expression levels remain unchanged, this is highly consistent with their roles as constitutive transcription factors regulated by post-transcriptional modification. indeed, this network includes dusp, a plasticity-related phosphatase and map3k, a kinase known to phosphorylate and activate nf-κb, as well as the nf-κb regulatory molecule, nf-κbid. together these data suggest that sappα treatment not only enhances the levels of transcription factors but also contributes to a dynamic interplay between a subset of protein kinases and phosphatases likely to maintain transcriptional activity. regulation of the chromatin environment is a third predicted mechanism by which sappα regulates expression of genes in this network. the network contains mediators of chromatin condensation acetyltransferase; + fold) and histone biotinylation ) ligase; + fold). upregulation of kat2b is likely to result in reduced condensation of chromatin, occurring via acetylation of tnp a molecule also regulated within this network. to further explore the min dataset we used the functional annotation tool, david. this highlighted cognition and sensory perception as well as response to corticosterone as significantly enriched functions. interestingly, there is an increase in basal glucocorticoid levels in alzheimer’s disease,. thus sappα may regulate the expression of genes associated with this pathway. sappα-induced regulation of apoptosis and the inflammatory response we next explored the gene networks affected by exposure to sappα for extended times. following h exposure to sappα, inflammatory response was the most significant biological function of the highest scoring network formed used ipa. this network contains the proinflammatory cytokine il1a, previously associated with alzheimer’s disease, development and plasticity, immune associated genes and chemokines. these chemokines are involved in chemoattraction of immune cells to sites of tissue damage but have widespread non-immunological effects in the central nervous system, including regulation of neural cell proliferation, migration, survival and synaptic transmission. that sappα treatment promotes regulation of chemokine responses is also supported from analysis of the h data set with the functional annotation tool, david. like the min network, nf-κb forms a central hub in this network, however, many of the genes contributing to the hub, including ehf, which promotes apoptosis, are downregulated. interestingly, using the upstream regulatory element analysis tool to analyse the genes upregulated within the h dataset, it is predicted that sappα induces inhibition of the transcription factor, tumour protein, which is also an important regulator of apoptosis, and is increased in alzheimer’s disease. in addition, upstream regulatory element analysis using the entire h dataset, predicts activation of the transcription factors ap- hmgb and stat suggesting other mechanisms by which sappα coordinates this gene response. sappα-induced long-term regulation of neurogenic, inflammatory response and cell survival pathways as the reported biological functions of sappα, such as neuroprotection and regulation of memory, are enduring physiological changes, we explored the gene expression response induced by a h exposure to sappα. to identify the functional relationships within the h differentially expressed gene list, we used the david functional annotation tool. this analysis identified neurogenesis, morphogenesis and development as important biological functions affected h post-sappα treatment. we extended analysis of this dataset by interpretation of the highest scoring network derived using ipa,. this network was composed of genes with overlapping biological functions, including upregulated genes related to cell proliferation, regulation of genes likely to promote cell survival and inhibit apoptosis. as sappα has previously been shown to promote growth, survival and proliferation in neurons and stem cells these findings supports a role for sappα-induced gene expression in mediating these events. discussion in order to better understand the role that regulation of gene expression may play in mediating the wide-ranging cellular effects of sappα, we performed a global transcriptome analysis in organotypic hippocampal slice cultures treated with nm sappα for min, h or h. we focused on the hippocampus as this region is essential to learning and memory and is especially vulnerable to degeneration in alzheimer’s disease. our analysis demonstrated that sappα rapidly regulates gene expression by engaging multiple transcriptional regulatory mechanisms. this is evidenced by rapid and transient upregulation of inducible transcription factors, temporally specific regulation of constitutively expressed transcription factors and microrna, as well as, modulation of the chromatin environment. network analysis showed that regulation of nf-κb is a consistent theme across all three time-points investigated. this supports previous work proposing that the neuroprotective effects of sappα are mediated through activation of nf-κb and subsequent enhanced activation of gene expression. sappα-induced nf-κb activation is implicated in protecting neural cells from apoptosis, therefore our results suggest a role for specifically regulated genes in this process. interestingly, nf-κb has recently been intimately linked with amyloid-ß production and sappα modulates app processing. therefore, the control of amyloid-β production may be crucially dependent on the activation status of nf-κb, over which sappα has influence. increasing evidence points to the involvement of dysregulated microrna in alzheimer’s disease and indeed an nf-κb-sensitive microrna has been implicated in modulating the inflammatory circuit in alzheimer’s disease. our results highlight a role for microrna in sappα-induced regulation of gene expression. we found that a short-term exposure to sappα was associated with upregulation of a subset of microrna. as medium to long-term sappα exposure resulted in a general decrease in gene expression, we propose that this may be mediated by microrna. interestingly, we reported a similar general effect on gene expression following the induction of ltp, which we have shown to be associated with altered levels of mature microrna. our results also demonstrate that sappα induces a subset of plasticity-associated immediate early genes, however, we found little evidence that the sappα-induced gene response paralleled that of the ltp gene response. therefore, post-translational modifications, and altered synaptic protein synthesis, are likely to mediate many of the effects of sappα on ltp. it is of particular note that prolonged exposure to sappα resulted in persistent effects on gene expression that correlate closely with the documented neuroprotective and neurogenic roles of sappα. this interpretation is consistent with the only other microarray analysis of sappα-regulated gene expression in the hippocampus. this study reported that treatment of mouse organotypic slices with nm sappα for h also resulted in regulation of several neuroprotective genes. interestingly, there was little coherence between the genes identified in this study and our own. this may result from differences in the array type used, the number of annotated genes investigated, the statistical analyses performed or species. while there is a paucity of data estimating the physiological levels of sappα, it has been estimated to fall within the pm range within plasma and brain homogenates, however, it is difficult to extrapolate these concentrations to the ex vivo model used in this study. our studies highlight that nm sappα is sufficient to modulate gene expression in ex vivo models and emphasizes that both medium and longer-term exposure to sappα elicits an inflammatory and immune gene response, which likely provides a neuroprotective environment. this neuroprotective setting appears to be strengthened by a parallel downregulation of apoptotic pathways and increases in cell proliferation and survival. furthermore, we found evidence for the regulation of genes associated with neurogenesis. recent evidence has also linked sappα with the induction of neurogenesis in the mammalian brain. as new brain cells have the capacity to integrate into previously established neural networks and contribute to hippocampal functioning, neurogenesis may also contribute to the sappα-induced neuroprotective and memory enhancing effects over the long-term.  in summary, our analyses consolidate the concept that sappα regulates gene expression. we provide evidence that this occurs in a temporally specific manner, and occurs through providing an environment conducive for transcription that results in activation of immediate early gene transcription factors, known to mediate neuroprotection and proliferation, and regulation of microrna. furthermore, we demonstrate that gene networks constructed following medium and prolonged exposure to sappα reveal novel mechanisms likely to underpin and consolidate the neuroprotective stimulus induced by sappα in the hippocampus. 
while these results suggest commonality between sappα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. our results demonstrate that sappα rapidly and persistently regulates gene expression in rat hippocampus. ingenuity pathway analysis predicted that short-term exposure to sappα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors, modulation of the chromatin environment, and apparent activation of the constitutive transcription factors creb and nf-κb. slices were exposed to nm sappα or phosphate buffered saline for min, h or h and sappα-associated gene expression profiles were produced for each time-point using affymetrix rat gene st arrays. treatment of organotypic hippocampal slice cultures with nm sappα induced temporally distinct gene expression profiles, including mrna and microrna associated with alzheimer’s disease. having demonstrated that treatment with human recombinant sappα was protective against n-methyl d-aspartate-induced toxicity, we next explored the sappα-induced gene expression profiles. as these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sappα in rat hippocampal organotypic slice cultures. in contrast, medium and long exposure to sappα resulted in an overall downregulation of gene expression. importantly, dynamic regulation of nf-κb appears to be integral to the transcriptional response across all time-points. the gene networks formed following medium and long exposure to sappα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sappα. differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of alzheimer’s disease, or secreted amyloid precursor protein-alpha, which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity.
 differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of alzheimer’s disease, or secreted amyloid precursor protein-alpha, which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. the underlying molecular mechanisms recruited by sappα that underpin these considerable cellular effects are not well elucidated. as these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sappα in rat hippocampal organotypic slice cultures. slices were exposed to nm sappα or phosphate buffered saline for min, h or h and sappα-associated gene expression profiles were produced for each time-point using affymetrix rat gene st arrays.  treatment of organotypic hippocampal slice cultures with nm sappα induced temporally distinct gene expression profiles, including mrna and microrna associated with alzheimer’s disease. having demonstrated that treatment with human recombinant sappα was protective against n-methyl d-aspartate-induced toxicity, we next explored the sappα-induced gene expression profiles. ingenuity pathway analysis predicted that short-term exposure to sappα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors, modulation of the chromatin environment, and apparent activation of the constitutive transcription factors creb and nf-κb. importantly, dynamic regulation of nf-κb appears to be integral to the transcriptional response across all time-points. in contrast, medium and long exposure to sappα resulted in an overall downregulation of gene expression. while these results suggest commonality between sappα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. the gene networks formed following medium and long exposure to sappα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sappα.  our results demonstrate that sappα rapidly and persistently regulates gene expression in rat hippocampus. this regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sappα. secreted amyloid precursor protein alphahippocampusorganotypic slice culturesmicroarrayingenuity pathway analysisneuroprotectionimmediate early genesmicrornanf-κb 