BACKGROUND
alzheimer’s disease  is an age-related neurodegenerative disease characterized by selective dysfunction and loss of neurons in specific regions of the brain including the cortex and hippocampus  <cit> .
the levels of neuronal dysfunction correlate with learning and memory deficits that severely impede a patient’s ability to live independently.
amyloid precursor protein  and its derivatives  are thought to play a central role in ad, affecting both intra- and extra-neuronal processes  <cit> .
with an aging population, ad is on the increase and could affect more than  <dig> million people worldwide by  <dig>  <cit> .
attempts to develop new treatments for ad have proven unsuccessful  <cit>  which likely highlights the lack of understanding of the disease, particularly its onset and early progression.
ad is commonly divided into two subtypes – early onset  and late onset   <cit> .
early-onset ad generally presents itself before age  <dig> with late-onset ad developing later in life.
mutations identified in genes such as app and presenilin  <dig> and  <dig>  contribute to early onset ad  <cit> .
variations in many genes including apolipoprotein e  have been associated with late-onset ad, but their interactions to cause ad are unclear  <cit> .
key hallmarks of both types of ad are neurofibrillary tangles and aβ plaques  <cit> .
however, their appearance, particularly neurofibrillary tangles, are likely to represent stages of the disease at which neuronal dysfunction has already begun, and it may be difficult to develop treatments that effectively target events from these stages onwards.
therefore it is essential to better understand the earlier stages of the disease that precede the onset of neurofibrillary tangles and plaques.
these earlier stages are likely to extend over many decades in humans and targeting them provides the greatest opportunity for therapeutic intervention.
identifying the onset and early progression of any complex age-related disease using humans or non-human primates alone is particularly challenging.
therefore, an increased understanding of the molecular and cellular processes occurring during early disease stages requires the use of animal models.
the mouse has been widely used to investigate sub-phenotypes of ad such as amyloidosis  <cit> , reactive astrogliosis  <cit>  and neuroinflammation  <cit> .
however, the mouse models have not been used to identify the molecular events that occur very early during disease, prior to or at the onset of the traditional hallmarks of ad.
to identify these early molecular changes we performed clustering of transcriptional profiling data generated from mouse brains that showed a range in disease severity from no to early signs of ad.
we had previously applied transcript clustering to identify early molecular stages in a mouse model of glaucoma  <cit> .
in this present study, we used the appswepsen1de <dig> mouse model of ad, which is reported to develop plaques from 6 months of age  <cit> .
transcript clustering identifies age-specific changes as critical for the onset and early progression of ad in these mice.
moreover, we used clustering of transcriptional profiles to identify changes to insulin signaling as an important early event in ad.
methods
mouse strains and husbandry
c57bl/6j.appswepsen1de <dig> mice  were obtained from the jackson laboratory and maintained in 14/10-hour light/dark cycle.
all experiments were approved by the animal care and use committee at the jackson laboratory.
to generate experimental mice, b <dig> apbtg/+ mice were mated to c57bl/6 j  mice to generate ad and wild-type  cohorts.
to minimize gene expression variation between mice, all mice in experimental cohorts were bred in the same mouse room, were aged together  and only females were assessed.
mice from 2–12 months old were used in this study.
tissue harvesting, rna isolation and sequencing
tissue harvesting
a total of  <dig> mice were selected for transcriptional profiling:  <dig> at 4 months ,  <dig> at 5 months  and  <dig> at 6 months .
at the above ages, mice were sacrificed and the brain dissected free from the skull.
the left hemisphere was snap frozen for preservation of rna for gene expression studies.
the right hemisphere was fixed in 4% paraformaldehyde overnight at 4°c and stored in 1× phosphate-buffered solution  at 4°c for future use.
rna isolation and library preparation
rna was extracted from the left hemisphere of each brain using trizol .
mrna was purified from total rna using biotin-tagged poly dt oligonucleotides and streptavidin-coated magnetic beads followed by qc using an agilent technologies  <dig> bioanalyzer.
the mrna was then fragmented, and double-stranded cdna generated by random priming.
the ends of the fragmented dna were converted into phosphorylated blunt ends.
an ‘a’ base was added to the 3’ ends.
illumina®-specific adaptors were ligated to the dna fragments.
using magnetic bead technology, the ligated fragments were size-selected and then a final pcr was performed to enrich the adapter-modified dna fragments, since only the dna fragments with adaptors at both ends will amplify.
high-throughput sequencing
to minimize sequencing batch effects, all  <dig> samples were barcoded and combined, and sequenced across six lanes on an illumina hiseq  <dig> using standard conditions to generate 100 bp paired end sequences.
a minimum of  <dig> million paired end reads were generated for each sample.
rna sequence analysis and clustering
analysis of sequence was performed in a private instance of the publicly available galaxy .
quality control of sequence
sequence quality was assessed using fastqc qc .
results showed that the first  <dig> bases showed minor sequencing bias so these were trimmed from the sequencing reads.
after trimming, the average quality score at each base position was greater than  <dig> .
alignment of sequence
tophat/bowtie  was used to align sequences to the mouse genome .
flagstat  was used to show that at least  <dig> % of pair end reads aligned to the mouse genome.
determining fpkm
for each sample, fragment length per kilobase of exon per million fragments mapped  values were generated using cufflinks .
quartile normalization  and bias correction  were used.
mouse transcripts were taken from the ncbim <dig>  gene set.
determining differentially expressed genes
differentially expressed genes were determined using cuffdiff .
again, quartile normalization and bias correction were used.
computational analyses
singular value decomposition and hierarchical clustering
for each data subset, aligned paired-end reads were filtered to remove any gene that was not expressed in any of the samples.
for the  <dig> samples at  <dig> and 6 months, filtering yielded  <dig> genes.
the profile of each gene was centered on the mean of wt samples at 4 months.
hierarchical clustering was performed based on euclidean distance between genes across all samples to identify overall similarity.
singular value decomposition  was used to identify common signals and verify consistency across replicate cohorts.
the first svd component of the  <dig> samples at  <dig> and 6 months, which separated the two age cohorts, accounted for 38% of the global variance.
the second component, primarily driven by mutant samples at 4 months, represented 10% of the variance.
for the subset of  <dig> samples taken at 4 months, the first svd component separated mutant from wt samples and accounted for 28% of the variance.
pathway analyses of differentially expressed genes
annotations were generated and overrepresented pathways were identified using david v <dig>  .
p values reported in this study were corrected for multiple tests.
pathways were colored using kyoto encyclopedia of genes and genomes .
immunofluorescence, immunohistochemistry and imaging
immunofluorescence
mice were transcardially perfused with 4% pfa in 1× pbs.
brains were dissected and postfixed in 4% pfa overnight, cryoprotected in 10% and 30% sucrose, and embedded in oct. frozen sections  were incubated for 1– <dig> nights in the following primary antibodies: mouse anti-phosphorylated neurofilament , rabbit anti-iba <dig> , rabbit anti-mbp , rabbit polyclonal gfap ; rabbit polyclonal oxt ; and goat polyclonal irs <dig> .
with the exception of anti-oxt, the rabbit polyclonal antibodies were diluted in pbtb  containing 10% of normal goat serum and the goat polyclonal antibody was diluted in pbt  containing 10% donkey serum.
for anti-oxt, the bsa was not included in the dilution buffer.
after incubation with the primary antibodies, the brain sections were washed with pbt and incubated in the respective secondary antibodies  for 2 h, washed in pbt, counterstained with dapi, and mounted with aqua-polymount.
for thio-t staining, brain sections processed for gfap immunohistochemistry were incubated 10 min in 1% thio-t , transferred to  <dig> % acetic acid for 10 min, rinsed in dh2o for 5 min and mounted with aqua-polymount.
thio-t is a dye that binds to beta sheet-rich structures such as amyloid plaques and produces similar results to those obtained with antibodies to aβ such as 4g <dig> and 6e <dig> .
imaging
for each antibody, at least 4 ad and  <dig> wt sections from multiple brain regions were assessed.
particular attention was given to the cortex, hippocampus and hypothalamus.
imaging and photography was performed on both a zeiss axio imager microscope and a leica sp <dig> confocal microscope.
post image processing was performed in fiji .
RESULTS
ad phenotypes first occur in b <dig> apbtg mice from 4 months of age
the main aim of this study was to use transcript profiling to identify molecular changes that impact the onset and very early stages of ad.
therefore, to determine the ideal ages to profile, we assessed b <dig> apbtg female mice between  <dig> and 12 months of age for ad phenotypes including plaque formation, reactive astrogliosis and activation of microglia .
previous reports using the b <dig> apbtg strain show that females develop plaques and other ad-like phenotypes from around 6–8 months of age  <cit> .
given the potential for environmental differences to influence onset and progression of ad in different colonies of mice, we first assessed ad phenotypes in our colony from 8–12 months of age.
as expected, mice of all ages within this span showed plaques in multiple brain regions and there was an observable increase in plaque numbers and size with increasing age .
activation of microglia both surrounding plaques and associated with axonal swellings was also observed at these time points .
as previously reported, we saw no significant loss of neurons in any brain regions including the cortex and hippocampus at these ages .
these results indicate that onset and early stages of ad are occurring in b <dig> apbtg female mice younger than 8 months old.
therefore, we assessed ad phenotypes in mice 2–6 months old.
at 2 months of age, b <dig> apbtg female mice were indistinguishable from wt controls.
however, by 4 months of age, very small plaques were observed in discrete regions of the brain, particularly in the cortex .
plaques were readily observed in 6 months mice in the cortex and hippocampus.
together these results indicated that profiling b <dig> apbtg female mice at 4–6 months of age would identify very early molecular changes that may be key drivers and/or important biomarkers of early stages of ad.
clustering of transcriptional profiles identifies age is a major driver of disease onset
to identify early molecular changes in ad, we used rna-seq to generate transcriptional profiles from  <dig> female mice  from  <dig>   <dig>  and 6 months of age .
for each sample, reads were aligned using tophat  <cit>  and normalized fpkm values were generated for every expressed gene using cufflinks  <cit> .
to determine the processes driving variation between all  <dig> and 6 months samples, we used singular value decomposition .
genes that were not expressed in any samples were removed from the analyses.
strikingly, the largest component of variation was age as the greatest separation was observed between the  <dig> and 6 months samples .
genotype  was the second major component .
hierarchical clustering of the samples showed a similar outcome .
therefore, key age-specific changes are likely to occurring at these ages to drive the onset and early progression of ad phenotypes.
to identify these age-specific changes responsible for the variation between the  <dig> and 6 months samples, we compared transcript profiles from all 4 months samples  to profiles from all 6 months samples  using cuffdiff .
a total of  <dig> genes were differentially expressed  between the ‘all 4 months’ and ‘all 6 months’ groups .
similar results were obtained when comparing either the 4 months ad samples to the 6 months ad samples, or the 4 months wt to the 6 months wt samples  indicating that these truly are age-specific changes.
pathway analysis revealed that the ribosome and oxidative phosphorylation pathways were over-represented in the  <dig> de genes .
these pathways have previously been linked to aging including down-regulation of protein synthesis/mrna processing and mitochondrial dysfunction  <cit> .
our data imply that perturbations in these pathways are necessary for the onset or early progression of ad in b <dig> apbtg mice.
understanding these age-specific changes is likely important for all forms of alzheimer’s disease, not just the familial form modeled in b <dig> apbtg mice.
disruption to genes normally associated with the hypothalamic-pituitary-adrenal  axis is an early event in b <dig> apbtg mice
after age, svd identifies ad genotype as the second greatest component of variation in the  <dig> samples from the 4 months group .
this is the age at which ad phenotypes were first observed .
however, hierarchical clustering identifies that  <dig> samples  are outliers and likely to decrease the sensitivity to detect true ad-relevant changes .
therefore, to identify de genes important for ad onset we compared the transcript profiles of the  <dig> most closely related 4 months samples  using cuffdiff.
a total of  <dig> genes were de between the 4 months ad and wt groups .
six of the most downregulated genes comparing the 4 months ad and wt groups are hormones or hormone-related.
these six genes include growth hormone , pro-opiomelanocortin  and oxytocin .
interestingly, these six genes are commonly associated with the hpa region of the brain and transcript analysis  shows expression for oxt and pomc only in the hypothalamus.
our data shows similar findings; with the oxt protein almost exclusively localized to the hypothalamic regions surrounding the third ventricle .
these hpa-associated genes are involved in a variety of different behaviors including depression, appetite and metabolism.
these behaviors have been previously associated with human alzheimer’s disease  <cit> .
our analyses indicate that alterations in these behaviors may occur early in ad and studying these genes may be important for developing new therapies or as diagnostic criteria for ad.
clustering of 5 months samples identifies perturbation in the insulin signaling pathway as an important event in ad
our combined approach of histology, transcriptional profiling and computational analyses demonstrates that key events, including age-specific changes, occur between  <dig> and 6 months of age in b <dig> apbtg mice.
therefore, we hypothesized that early progression of ad is likely to be occurring at 5 months as well as at 4 months of age in these mice.
to test this, we analyzed the transcriptional profiles from the  <dig> ad transgene carrying b <dig> apbtg female mice at 5 months of age.
we first determined the similarity of these transcriptional profiles to the  <dig> and 6 months ad and wt samples using hierarchical clustering.
rather than clustering into a separate ‘age-specific’ group, the 5 months samples clustered with the  <dig> and 6 months samples: three of the seven 5 months ad samples clustered with the 4 months samples and four of the 5 months samples clustered with the 6 months samples .
this shows that indeed crucial changes are occurring independent of absolute age in ad mice at 5 months of age.
these changes may be ‘age-specific’ due to the 5 months old mice aging at different rates , or ‘disease-specific’ changes independent of age, or both.
to help answer these questions and to determine the gene expression differences driving the variation between the two groups of 5 months samples  we used cuffdiff.
a total of  <dig> genes were differentially expressed comparing the early to late group .
pathway analysis of the de genes identified the ribosome pathway and the insulin signaling pathway as overrepresented in stage  <dig> compared to stage  <dig> .
the ribosome pathway was also overrepresented comparing the  <dig> to the 6 months data suggesting that differences in physiological aging are driving some of the variation between the chronologically-matched 5 months old samples.
insulin signaling is a novel finding that was only significant in clustering of the 5 months samples.
therefore, it is not clear whether the variation between the 5 months old samples is due in part to the mice physiologically aging at different rates, or disease-specific changes.
at least six genes in the insulin signaling pathway are differentially expressed in stage  <dig> compared to stage  <dig> .
these include three upregulated genes  and three downregulated genes .
insulin receptor substrate  <dig>  is the most downregulated gene in the insulin signaling pathway and is of particular interest as irs genes have been shown to impact phosphorylation of tau, a crucial step in the generation of neurofibrillary tangles  <cit> .
previous studies have localized irs <dig> to specific sets of neurons in the hypothalamus  <cit>  and allen brain atlas for mouse shows expression of irs <dig> mainly in the hypothalamus and at lower levels in pallidum.
interestingly, the human allen brain reports expression of irs <dig> in both the hypothalamus and cortex.
our analysis of irs <dig> expression agrees with these findings with strongest expression in the hypothalamus and possible low-level expression in the cortex .
to confirm this we localized the irs <dig> protein using an anti-irs <dig> antibody .
irs <dig> is indeed present in axons of neurons in both the hypothalamus and the cortex .
given our previous findings of early changes to genes associated with the hypothalamus, it is possible that the downregulation of irs <dig>  is occurring in the hpa region, but it is also possible changes are occurring in the cortex directly and these may be key to the onset/progression of alzheimer’s disease in these mice.
discussion and
CONCLUSIONS
here we report the use of transcript clustering to identify molecular mechanisms contributing to early stages of ad in mice.
this is the first time this approach has been used to assess early events in ad.
we show that plaques first appear in the cortex of female b <dig> apbtg mice around 4 months, and more significantly at 6 months of age.
transcript clustering identified age as the greatest driver of variation in the  <dig> and 6 months samples, with genotype being the second major driver.
pathway analysis of the genes differentially expressed between the  <dig> and 6 months samples suggests at least two biological processes are driving these changes namely mrna processing/protein translation and oxidative phosphorylation.
both of these processes have previously been implicated in age-related changes  <cit> .
although it is unexpected that these aging-linked changes occur so early, it may suggest that subtle aging changes occur in mice as early as 4–6 months.
at this age, mice are considered to be equivalent in age to early adulthood in humans  an age when mild cognitive impairment  can occur in some individuals  <cit> .
the earliest ad-relevant changes detected in 4 months ad mice compared to wt mice were hormone-related and may indicate changes to the hpa axis.
our study is the first to propose that these may be among the earliest changes in ad and that they may be important indicators of susceptibility to and the onset of ad.
genes expressed by cells in the hpa axis control behaviors such as mood, sleep, and eating and these behaviors are commonly disrupted in ad patients.
for instance, systemic metabolic changes as well as mood changes have been observed in very early phases of ad and suggest early dysfunction in the hypothalamus  <cit> .
another change seen in ad patients is a behavioral one, affecting mood and depression  <cit> .
oxytocin, often prescribed as a medication to treat depression, is almost exclusively expressed in the hypothalamus  <cit>  and our data shows a downregulation early in b <dig> apbtg mice.
it is not clear why the hypothalamus may be more susceptible than other brain regions to early dysfunction in ad but it may be due to the absence of a traditional blood brain barrier in this region.
instead, vessels surrounding the hypothalamus, particularly the median basal region of the hypothalamus, are fenestrated to allow the easy passage of hormones into the circulation  <cit> .
this structure is constitutively leaky and may be particularly susceptible to increases in ad-relevant phenotypes such as amyloid accumulation.
furthermore, vascular dysfunction  may precede the molecular changes identified in our study.
age-related vascular dysfunction has been reported but more detailed studies relating to the role of age-related vasculature dysfunction in ad are required.
transcript clustering of the 5 months data identified changes to the insulin signaling pathway including irs <dig> as an early event in ad.
insulin signaling is strongly associated with aging and age-related diseases including obesity and diabetes, conditions that have recently been identified as possible risk factors for ad  <cit> .
irs <dig> is expressed strongly in the hypothalamus but at lower levels in other brain regions such as the cortex.
therefore, downregulation of irs <dig> likely impacts both the hypothalamus and the cortex.
irs <dig> is a good candidate for further studies as it is known to function in a number of processes relevant to ad including interacting with endosomes  <cit>  to control aβ levels in neurons  <cit> , or through interactions with irs <dig> <cit>  in the phosphorylation of tau during the formation of neurofibrillary tangles  <cit> .
although further work is required to elucidate the precise role of irs <dig> in ad, our data suggests that understanding the role of irs <dig> in ad may lead to potential new therapies for ad.
our study, using a genetic model relevant to early onset ad, shows that clustering of transcriptional profiling data is a powerful method to identify important early molecular changes at the onset of traditional hallmarks of ad.
however, it is widely accepted that these models do not recapitulate all aspects of ad including hyperphosphorylation of tau leading to neurofibrillary tangles and neuronal cell loss.
this is intriguing given that mutations that disrupt app processing do cause neurofibrillary tangles and associated neuronal changes in human ad.
given this, our work highlights the need for further investigation of b <dig> apbtg mice at the onset of plaque formation to better understand the role of insulin signaling and other early molecular events that follow app misprocessing.
furthermore, our study focuses on a mouse model relevant to early onset ad, which accounts for <5% of all cases human of ad.
it is not clear whether mechanisms that lead to plaque formation and neurofibrillary tangles in early onset ad are similar or distinct to those that lead to ad in late-onset ad.
nevertheless, understanding and targeting these early events is likely to provide the greatest potential for developing new treatments for alzheimer’s disease.
availability of supporting data
raw data is being made available through geo datasets.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
grh designed and implemented the study.
hmj, is and lcg performed the histological analyses.
hmg and grh performed the rna-seq data analyses.
gwc performed the computational analyses including singular value decomposition and hierarchical clustering.
grh wrote the manuscript and it was critically read and approved by all authors.
supplementary material
additional file 1: table s1
differentially expressed genes comparing  <dig> months with  <dig> months samples.
click here for file
additional file 2: table s2
differentially expressed genes comparing  <dig> months wt with  <dig> months ad samples.
click here for file
additional file 3: table s3
differentially expressed genes comparing stage  <dig> with stage  <dig> samples.
click here for file
acknowledgements
this work was supported in part by the jane b. cook  <dig> charitable trust.
g.w.c.
was supported in part by grant k <dig> gm <dig> from nigms.
we thank katherine macnicoll and sam groh for mouse colony maintenance and tissue harvesting and acknowledge the scientific services at the jackson laboratory including genome technologies, computational sciences and imaging sciences.
we are grateful to stephen sampson for careful reading of the manuscript.

 alzheimer’s disease is an age-related neurodegenerative disease characterized by selective dysfunction and loss of neurons in specific regions of the brain including the cortex and hippocampus. the levels of neuronal dysfunction correlate with learning and memory deficits that severely impede a patient’s ability to live independently. amyloid precursor protein and its derivatives are thought to play a central role in ad, affecting both intra- and extra-neuronal processes. with an aging population, ad is on the increase and could affect more than million people worldwide by. attempts to develop new treatments for ad have proven unsuccessful which likely highlights the lack of understanding of the disease, particularly its onset and early progression. ad is commonly divided into two subtypes – early onset and late onset. early-onset ad generally presents itself before age with late-onset ad developing later in life. mutations identified in genes such as app and presenilin and contribute to early onset ad. variations in many genes including apolipoprotein e have been associated with late-onset ad, but their interactions to cause ad are unclear. key hallmarks of both types of ad are neurofibrillary tangles and aβ plaques. however, their appearance, particularly neurofibrillary tangles, are likely to represent stages of the disease at which neuronal dysfunction has already begun, and it may be difficult to develop treatments that effectively target events from these stages onwards. therefore it is essential to better understand the earlier stages of the disease that precede the onset of neurofibrillary tangles and plaques. these earlier stages are likely to extend over many decades in humans and targeting them provides the greatest opportunity for therapeutic intervention. identifying the onset and early progression of any complex age-related disease using humans or non-human primates alone is particularly challenging. therefore, an increased understanding of the molecular and cellular processes occurring during early disease stages requires the use of animal models. the mouse has been widely used to investigate sub-phenotypes of ad such as amyloidosis, reactive astrogliosis and neuroinflammation. however, the mouse models have not been used to identify the molecular events that occur very early during disease, prior to or at the onset of the traditional hallmarks of ad. to identify these early molecular changes we performed clustering of transcriptional profiling data generated from mouse brains that showed a range in disease severity from no to early signs of ad. we had previously applied transcript clustering to identify early molecular stages in a mouse model of glaucoma. in this present study, we used the appswepsen1de mouse model of ad, which is reported to develop plaques from 6 months of age. transcript clustering identifies age-specific changes as critical for the onset and early progression of ad in these mice. moreover, we used clustering of transcriptional profiles to identify changes to insulin signaling as an important early event in ad. methods mouse strains and husbandry c57bl/6j.appswepsen1de mice were obtained from the jackson laboratory and maintained in 14/10-hour light/dark cycle. all experiments were approved by the animal care and use committee at the jackson laboratory. to generate experimental mice, b apbtg/+ mice were mated to c57bl/6 j mice to generate ad and wild-type cohorts. to minimize gene expression variation between mice, all mice in experimental cohorts were bred in the same mouse room, were aged together and only females were assessed. mice from 2–12 months old were used in this study. tissue harvesting, rna isolation and sequencing tissue harvesting a total of mice were selected for transcriptional profiling: at 4 months, at 5 months and at 6 months. at the above ages, mice were sacrificed and the brain dissected free from the skull. the left hemisphere was snap frozen for preservation of rna for gene expression studies. the right hemisphere was fixed in 4% paraformaldehyde overnight at 4°c and stored in 1× phosphate-buffered solution at 4°c for future use. rna isolation and library preparation rna was extracted from the left hemisphere of each brain using trizol. mrna was purified from total rna using biotin-tagged poly dt oligonucleotides and streptavidin-coated magnetic beads followed by qc using an agilent technologies bioanalyzer. the mrna was then fragmented, and double-stranded cdna generated by random priming. the ends of the fragmented dna were converted into phosphorylated blunt ends. an ‘a’ base was added to the 3’ ends. illumina®-specific adaptors were ligated to the dna fragments. using magnetic bead technology, the ligated fragments were size-selected and then a final pcr was performed to enrich the adapter-modified dna fragments, since only the dna fragments with adaptors at both ends will amplify. high-throughput sequencing to minimize sequencing batch effects, all samples were barcoded and combined, and sequenced across six lanes on an illumina hiseq using standard conditions to generate 100 bp paired end sequences. a minimum of million paired end reads were generated for each sample. rna sequence analysis and clustering analysis of sequence was performed in a private instance of the publicly available galaxy. quality control of sequence sequence quality was assessed using fastqc qc. results showed that the first bases showed minor sequencing bias so these were trimmed from the sequencing reads. after trimming, the average quality score at each base position was greater than. alignment of sequence tophat/bowtie was used to align sequences to the mouse genome. flagstat was used to show that at least % of pair end reads aligned to the mouse genome. determining fpkm for each sample, fragment length per kilobase of exon per million fragments mapped values were generated using cufflinks. quartile normalization and bias correction were used. mouse transcripts were taken from the ncbim gene set. determining differentially expressed genes differentially expressed genes were determined using cuffdiff. again, quartile normalization and bias correction were used. computational analyses singular value decomposition and hierarchical clustering for each data subset, aligned paired-end reads were filtered to remove any gene that was not expressed in any of the samples. for the samples at and 6 months, filtering yielded genes. the profile of each gene was centered on the mean of wt samples at 4 months. hierarchical clustering was performed based on euclidean distance between genes across all samples to identify overall similarity. singular value decomposition was used to identify common signals and verify consistency across replicate cohorts. the first svd component of the samples at and 6 months, which separated the two age cohorts, accounted for 38% of the global variance. the second component, primarily driven by mutant samples at 4 months, represented 10% of the variance. for the subset of samples taken at 4 months, the first svd component separated mutant from wt samples and accounted for 28% of the variance. pathway analyses of differentially expressed genes annotations were generated and overrepresented pathways were identified using david v. p values reported in this study were corrected for multiple tests. pathways were colored using kyoto encyclopedia of genes and genomes. immunofluorescence, immunohistochemistry and imaging immunofluorescence mice were transcardially perfused with 4% pfa in 1× pbs. brains were dissected and postfixed in 4% pfa overnight, cryoprotected in 10% and 30% sucrose, and embedded in oct. frozen sections were incubated for 1– nights in the following primary antibodies: mouse anti-phosphorylated neurofilament, rabbit anti-iba, rabbit anti-mbp, rabbit polyclonal gfap; rabbit polyclonal oxt; and goat polyclonal irs. with the exception of anti-oxt, the rabbit polyclonal antibodies were diluted in pbtb containing 10% of normal goat serum and the goat polyclonal antibody was diluted in pbt containing 10% donkey serum. for anti-oxt, the bsa was not included in the dilution buffer. after incubation with the primary antibodies, the brain sections were washed with pbt and incubated in the respective secondary antibodies for 2 h, washed in pbt, counterstained with dapi, and mounted with aqua-polymount. for thio-t staining, brain sections processed for gfap immunohistochemistry were incubated 10 min in 1% thio-t, transferred to % acetic acid for 10 min, rinsed in dh2o for 5 min and mounted with aqua-polymount. thio-t is a dye that binds to beta sheet-rich structures such as amyloid plaques and produces similar results to those obtained with antibodies to aβ such as 4g and 6e. imaging for each antibody, at least 4 ad and wt sections from multiple brain regions were assessed. particular attention was given to the cortex, hippocampus and hypothalamus. imaging and photography was performed on both a zeiss axio imager microscope and a leica sp confocal microscope. post image processing was performed in fiji.  ad phenotypes first occur in b apbtg mice from 4 months of age the main aim of this study was to use transcript profiling to identify molecular changes that impact the onset and very early stages of ad. therefore, to determine the ideal ages to profile, we assessed b apbtg female mice between and 12 months of age for ad phenotypes including plaque formation, reactive astrogliosis and activation of microglia. previous reports using the b apbtg strain show that females develop plaques and other ad-like phenotypes from around 6–8 months of age. given the potential for environmental differences to influence onset and progression of ad in different colonies of mice, we first assessed ad phenotypes in our colony from 8–12 months of age. as expected, mice of all ages within this span showed plaques in multiple brain regions and there was an observable increase in plaque numbers and size with increasing age. activation of microglia both surrounding plaques and associated with axonal swellings was also observed at these time points. as previously reported, we saw no significant loss of neurons in any brain regions including the cortex and hippocampus at these ages. these results indicate that onset and early stages of ad are occurring in b apbtg female mice younger than 8 months old. therefore, we assessed ad phenotypes in mice 2–6 months old. at 2 months of age, b apbtg female mice were indistinguishable from wt controls. however, by 4 months of age, very small plaques were observed in discrete regions of the brain, particularly in the cortex. plaques were readily observed in 6 months mice in the cortex and hippocampus. together these results indicated that profiling b apbtg female mice at 4–6 months of age would identify very early molecular changes that may be key drivers and/or important biomarkers of early stages of ad. clustering of transcriptional profiles identifies age is a major driver of disease onset to identify early molecular changes in ad, we used rna-seq to generate transcriptional profiles from female mice from and 6 months of age. for each sample, reads were aligned using tophat and normalized fpkm values were generated for every expressed gene using cufflinks. to determine the processes driving variation between all and 6 months samples, we used singular value decomposition. genes that were not expressed in any samples were removed from the analyses. strikingly, the largest component of variation was age as the greatest separation was observed between the and 6 months samples. genotype was the second major component. hierarchical clustering of the samples showed a similar outcome. therefore, key age-specific changes are likely to occurring at these ages to drive the onset and early progression of ad phenotypes. to identify these age-specific changes responsible for the variation between the and 6 months samples, we compared transcript profiles from all 4 months samples to profiles from all 6 months samples using cuffdiff. a total of genes were differentially expressed between the ‘all 4 months’ and ‘all 6 months’ groups. similar results were obtained when comparing either the 4 months ad samples to the 6 months ad samples, or the 4 months wt to the 6 months wt samples indicating that these truly are age-specific changes. pathway analysis revealed that the ribosome and oxidative phosphorylation pathways were over-represented in the de genes. these pathways have previously been linked to aging including down-regulation of protein synthesis/mrna processing and mitochondrial dysfunction. our data imply that perturbations in these pathways are necessary for the onset or early progression of ad in b apbtg mice. understanding these age-specific changes is likely important for all forms of alzheimer’s disease, not just the familial form modeled in b apbtg mice. disruption to genes normally associated with the hypothalamic-pituitary-adrenal axis is an early event in b apbtg mice after age, svd identifies ad genotype as the second greatest component of variation in the samples from the 4 months group. this is the age at which ad phenotypes were first observed. however, hierarchical clustering identifies that samples are outliers and likely to decrease the sensitivity to detect true ad-relevant changes. therefore, to identify de genes important for ad onset we compared the transcript profiles of the most closely related 4 months samples using cuffdiff. a total of genes were de between the 4 months ad and wt groups. six of the most downregulated genes comparing the 4 months ad and wt groups are hormones or hormone-related. these six genes include growth hormone, pro-opiomelanocortin and oxytocin. interestingly, these six genes are commonly associated with the hpa region of the brain and transcript analysis shows expression for oxt and pomc only in the hypothalamus. our data shows similar findings; with the oxt protein almost exclusively localized to the hypothalamic regions surrounding the third ventricle. these hpa-associated genes are involved in a variety of different behaviors including depression, appetite and metabolism. these behaviors have been previously associated with human alzheimer’s disease. our analyses indicate that alterations in these behaviors may occur early in ad and studying these genes may be important for developing new therapies or as diagnostic criteria for ad. clustering of 5 months samples identifies perturbation in the insulin signaling pathway as an important event in ad our combined approach of histology, transcriptional profiling and computational analyses demonstrates that key events, including age-specific changes, occur between and 6 months of age in b apbtg mice. therefore, we hypothesized that early progression of ad is likely to be occurring at 5 months as well as at 4 months of age in these mice. to test this, we analyzed the transcriptional profiles from the ad transgene carrying b apbtg female mice at 5 months of age. we first determined the similarity of these transcriptional profiles to the and 6 months ad and wt samples using hierarchical clustering. rather than clustering into a separate ‘age-specific’ group, the 5 months samples clustered with the and 6 months samples: three of the seven 5 months ad samples clustered with the 4 months samples and four of the 5 months samples clustered with the 6 months samples. this shows that indeed crucial changes are occurring independent of absolute age in ad mice at 5 months of age. these changes may be ‘age-specific’ due to the 5 months old mice aging at different rates, or ‘disease-specific’ changes independent of age, or both. to help answer these questions and to determine the gene expression differences driving the variation between the two groups of 5 months samples we used cuffdiff. a total of genes were differentially expressed comparing the early to late group. pathway analysis of the de genes identified the ribosome pathway and the insulin signaling pathway as overrepresented in stage compared to stage. the ribosome pathway was also overrepresented comparing the to the 6 months data suggesting that differences in physiological aging are driving some of the variation between the chronologically-matched 5 months old samples. insulin signaling is a novel finding that was only significant in clustering of the 5 months samples. therefore, it is not clear whether the variation between the 5 months old samples is due in part to the mice physiologically aging at different rates, or disease-specific changes. at least six genes in the insulin signaling pathway are differentially expressed in stage compared to stage. these include three upregulated genes and three downregulated genes. insulin receptor substrate is the most downregulated gene in the insulin signaling pathway and is of particular interest as irs genes have been shown to impact phosphorylation of tau, a crucial step in the generation of neurofibrillary tangles. previous studies have localized irs to specific sets of neurons in the hypothalamus and allen brain atlas for mouse shows expression of irs mainly in the hypothalamus and at lower levels in pallidum. interestingly, the human allen brain reports expression of irs in both the hypothalamus and cortex. our analysis of irs expression agrees with these findings with strongest expression in the hypothalamus and possible low-level expression in the cortex. to confirm this we localized the irs protein using an anti-irs antibody. irs is indeed present in axons of neurons in both the hypothalamus and the cortex. given our previous findings of early changes to genes associated with the hypothalamus, it is possible that the downregulation of irs is occurring in the hpa region, but it is also possible changes are occurring in the cortex directly and these may be key to the onset/progression of alzheimer’s disease in these mice. discussion and  here we report the use of transcript clustering to identify molecular mechanisms contributing to early stages of ad in mice. this is the first time this approach has been used to assess early events in ad. we show that plaques first appear in the cortex of female b apbtg mice around 4 months, and more significantly at 6 months of age. transcript clustering identified age as the greatest driver of variation in the and 6 months samples, with genotype being the second major driver. pathway analysis of the genes differentially expressed between the and 6 months samples suggests at least two biological processes are driving these changes namely mrna processing/protein translation and oxidative phosphorylation. both of these processes have previously been implicated in age-related changes. although it is unexpected that these aging-linked changes occur so early, it may suggest that subtle aging changes occur in mice as early as 4–6 months. at this age, mice are considered to be equivalent in age to early adulthood in humans an age when mild cognitive impairment can occur in some individuals. the earliest ad-relevant changes detected in 4 months ad mice compared to wt mice were hormone-related and may indicate changes to the hpa axis. our study is the first to propose that these may be among the earliest changes in ad and that they may be important indicators of susceptibility to and the onset of ad. genes expressed by cells in the hpa axis control behaviors such as mood, sleep, and eating and these behaviors are commonly disrupted in ad patients. for instance, systemic metabolic changes as well as mood changes have been observed in very early phases of ad and suggest early dysfunction in the hypothalamus. another change seen in ad patients is a behavioral one, affecting mood and depression. oxytocin, often prescribed as a medication to treat depression, is almost exclusively expressed in the hypothalamus and our data shows a downregulation early in b apbtg mice. it is not clear why the hypothalamus may be more susceptible than other brain regions to early dysfunction in ad but it may be due to the absence of a traditional blood brain barrier in this region. instead, vessels surrounding the hypothalamus, particularly the median basal region of the hypothalamus, are fenestrated to allow the easy passage of hormones into the circulation. this structure is constitutively leaky and may be particularly susceptible to increases in ad-relevant phenotypes such as amyloid accumulation. furthermore, vascular dysfunction may precede the molecular changes identified in our study. age-related vascular dysfunction has been reported but more detailed studies relating to the role of age-related vasculature dysfunction in ad are required. transcript clustering of the 5 months data identified changes to the insulin signaling pathway including irs as an early event in ad. insulin signaling is strongly associated with aging and age-related diseases including obesity and diabetes, conditions that have recently been identified as possible risk factors for ad. irs is expressed strongly in the hypothalamus but at lower levels in other brain regions such as the cortex. therefore, downregulation of irs likely impacts both the hypothalamus and the cortex. irs is a good candidate for further studies as it is known to function in a number of processes relevant to ad including interacting with endosomes to control aβ levels in neurons, or through interactions with irs in the phosphorylation of tau during the formation of neurofibrillary tangles. although further work is required to elucidate the precise role of irs in ad, our data suggests that understanding the role of irs in ad may lead to potential new therapies for ad. our study, using a genetic model relevant to early onset ad, shows that clustering of transcriptional profiling data is a powerful method to identify important early molecular changes at the onset of traditional hallmarks of ad. however, it is widely accepted that these models do not recapitulate all aspects of ad including hyperphosphorylation of tau leading to neurofibrillary tangles and neuronal cell loss. this is intriguing given that mutations that disrupt app processing do cause neurofibrillary tangles and associated neuronal changes in human ad. given this, our work highlights the need for further investigation of b apbtg mice at the onset of plaque formation to better understand the role of insulin signaling and other early molecular events that follow app misprocessing. furthermore, our study focuses on a mouse model relevant to early onset ad, which accounts for <5% of all cases human of ad. it is not clear whether mechanisms that lead to plaque formation and neurofibrillary tangles in early onset ad are similar or distinct to those that lead to ad in late-onset ad. nevertheless, understanding and targeting these early events is likely to provide the greatest potential for developing new treatments for alzheimer’s disease. availability of supporting data raw data is being made available through geo datasets. competing interests the authors declare that they have no competing interests. authors’ contributions grh designed and implemented the study. hmj, is and lcg performed the histological analyses. hmg and grh performed the rna-seq data analyses. gwc performed the computational analyses including singular value decomposition and hierarchical clustering. grh wrote the manuscript and it was critically read and approved by all authors. supplementary material additional file 1: table s1 differentially expressed genes comparing months with months samples. click here for file additional file 2: table s2 differentially expressed genes comparing months wt with months ad samples. click here for file additional file 3: table s3 differentially expressed genes comparing stage with stage samples. click here for file acknowledgements this work was supported in part by the jane b. cook charitable trust. g.w.c. was supported in part by grant k gm from nigms. we thank katherine macnicoll and sam groh for mouse colony maintenance and tissue harvesting and acknowledge the scientific services at the jackson laboratory including genome technologies, computational sciences and imaging sciences. we are grateful to stephen sampson for careful reading of the manuscript.
alzheimer’s disease affects more than million people worldwide but there is no known cure. to identify these early events we used transcriptional profiling of mouse brains combined with computational approaches including singular value decomposition and hierarchical clustering. a combination of transcriptional profiling combined with computational analyses has uncovered novel features relevant to alzheimer’s disease in a widely used mouse model and offers avenues for further exploration into early stages of ad. our study identifies three key events in early stages of alzheimer’s disease. here, we used a mouse model of alzheimer’s disease to identify age-specific changes that occur prior to and at the onset of traditional alzheimer-related phenotypes including amyloid plaque formation. first, the most important drivers of alzheimer’s disease onset in these mice are age-specific changes. finally, insulin signaling, in particular the down-regulation of the insulin receptor substrate gene, may be an important event in the transition from age-related changes to alzheimer’s disease specific-changes. these include perturbations of the ribosome and oxidative phosphorylation pathways.
 alzheimer’s disease affects more than million people worldwide but there is no known cure. age is the strongest risk factor for alzheimer’s disease but it is not clear how age-related changes impact the disease. here, we used a mouse model of alzheimer’s disease to identify age-specific changes that occur prior to and at the onset of traditional alzheimer-related phenotypes including amyloid plaque formation. to identify these early events we used transcriptional profiling of mouse brains combined with computational approaches including singular value decomposition and hierarchical clustering.  our study identifies three key events in early stages of alzheimer’s disease. first, the most important drivers of alzheimer’s disease onset in these mice are age-specific changes. these include perturbations of the ribosome and oxidative phosphorylation pathways. second, the earliest detectable disease-specific changes occur to genes commonly associated with the hypothalamic-adrenal-pituitary axis. these include the down-regulation of genes relating to metabolism, depression and appetite. finally, insulin signaling, in particular the down-regulation of the insulin receptor substrate gene, may be an important event in the transition from age-related changes to alzheimer’s disease specific-changes.  a combination of transcriptional profiling combined with computational analyses has uncovered novel features relevant to alzheimer’s disease in a widely used mouse model and offers avenues for further exploration into early stages of ad. 