Abstract
Alzheimer’s disease (AD) and age-related cognitive decline represent a growing health burden and involve the hippocampus, a vulnerable brain region implicated in learning and memory. To understand the molecular effects of aging on the hippocampus, this study characterized the gene expression changes associated with aging in rodents using RNA-sequencing (RNA-seq). The glutamate modulator, riluzole, which was recently shown to improve memory performance in aged rats, prevented many of the hippocampal age-related gene expression changes. A comparison of the effects of riluzole in rats against human AD data sets revealed that many of the gene changes in AD are reversed by riluzole. Expression changes identified by RNA-Seq were validated by qRT–PCR open arrays. Riluzole is known to increase the glutamate transporter EAAT2’s ability to scavenge excess glutamate, regulating synaptic transmission. RNA-seq and immunohistochemistry confirmed an increase in EAAT2 expression in hippocampus, identifying a possible mechanism underlying the improved memory function after riluzole treatment.
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Introduction
Aging is associated with cognitive decline in humans, which impairs quality of life and contributes significantly to health-care costs.1 Similar declines are also observed in rodents and non-human primates.2 Aging is the primary risk factor for the dementia of Alzheimer’s disease (AD); and with significant increases in life expectancy, the prevalence of AD and age-related cognitive disorders is rising.3 The neural circuits affected in aging and AD are similar, involving the glutamatergic connections between cortical areas and with the hippocampal formation, a brain region in the medial temporal lobe that is critical for memory formation.4, 5, 6 The glutamatergic pyramidal neurons of the hippocampus are highly vulnerable to damage in both age-related cognitive decline and in AD.5, 7 However, the effects of glutamatergic modulation on aging remains unknown.
Riluzole is a glutamate modulator approved for treatment of amyotrophic lateral sclerosis.8 Importantly, riluzole treatment for 4 months prevented age-related cognitive decline in rodents through clustering of dendritic spines,9 which form the postsynaptic component of most excitatory synapses.10 Clustering of synaptic inputs is an important neuroplastic mechanism that increases synaptic strength, empowering neural circuits.11, 12 Riluzole’s ability to induce dendritic spines clustering,9 which is dependent on glutamatergic neuronal activity13, 14 and long-term potentiation (LTP),15 suggests that it regulates synaptic glutamatergic activity and prevents glutamate overflow to the extrasynaptic space. Synaptic N-methyl-D-aspartate (NMDA) activity is critical for LTP and memory formation, whereas extrasynaptic NMDA activation is associated with long-term depression (LTD) and excitotoxicity.16, 17, 18
The excitatory amino acid transporter 2 (EAAT2 or GLT-1; Slc1a2) is a high-affinity, Na+-dependent glutamate transporter and the dominant glutamate transporter in the brain.19, 20 Glutamate transporters, including EAAT2, decrease in aging21, 22 and AD,23, 24 and are associated with neurodegeneration.24 They also have a critical role in determining synaptic and extrasynaptic glutamate levels,20, 25 regulating physiological glutamatergic neurotransmission. Riluzole can act to stabilize the inactivated state of the voltage-gated sodium channel and it can increase EAAT2 expression,22, 26, 27, 28 potentiating glutamate uptake.28, 29, 30
Understanding the molecular vulnerabilities of glutamatergic neural circuits can point to novel and more effective treatment targets. In addition, molecular changes resulting from treatments that prevent cognitive decline remain largely unexplored. This study uses the combination of RNA-sequencing (RNA-seq) and open arrays to detect and validate specific molecular pathways that are changed by aging and with riluzole. Importantly, gene expression changes associated with a rescue of cognitive decline under a therapeutic intervention (riluzole) are identified. In addition, genes modulated by riluzole in the rat hippocampus are enriched in many of the same pathways, and in the opposite direction, as those altered in human AD gene expression data sets. These molecular transcriptional profiles in the aging hippocampus and with glutamatergic modulation by riluzole provide mechanistic insights into age-related cognitive decline and provide support for future studies on the role of glutamate transporters as potential therapeutic targets in the aging brain.
Materials and methods
Animals
Young (3-month-old) and aged male Sprague–Dawley rats (retired breeders, 10 months old, Harlan Laboratories (Harlan Sprague Dawley, Indianapolis, IN, USA) were housed at Rockefeller University for the duration of the experiments. All rats were pair-housed in climate-controlled conditions (30–50% humidity, 21±2 °C, 12 h-light/-dark cycle). Separate cohorts of animals were used for RNA extraction and immunohistochemical experiments. For RNA-seq experiments, each group had n=6: 3-month-old (young rats), 10-month-old (middle age rats), 14-month-old riluzole-treated rats and 14-month-old riluzole untreated rats. For immunohistochemistry, 3-month-old rats (n=10), 14-month-old untreated rats (n=9) and 14-month-old riluzole-treated rats (n=10) were used. All procedures were in agreement with the National Institutes of Health and The Rockefeller University Institutional Animal Care and Use Committee guidelines. Sample sizes were chosen to minimize the number of animals used given previously published reports using these methodologies.31, 32, 33
Riluzole treatment
Treated rats had ad libitum access to riluzole solution from 10 months to 14 months of age (17 weeks), and aged-control and young-control rats had ad libitum access to tap water. All rats had ad libitum access to food. The riluzole compound (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in tap water at a concentration of 110 μg ml−1, translating to ∼4.0 mg kg−1 per day per os. To make the solution, riluzole was stirred in room temperature tap water for 6 h. All containers with riluzole were covered with foil to prevent light exposure. Fresh solutions were made every 2–3 days for the duration of treatment.
Tissue processing and immunohistochemistry
One week after the end of riluzole treatment, rats were deeply anesthetized with 100 mg kg−1 sodium pentobarbital and transcardially perfused with 1.0% paraformaldehyde in 0.1 M phosphate buffer (PB; 1 min) followed by 4.0% paraformaldehyde+0.125% glutaraldehyde in 0.1 M PB (12 min). Brains were removed and post-fixed for 6 h in 4.0% paraformaldehyde+0.125% glutaraldehyde in 0.1 M PB (4°C) and transferred to 0.1% sodium azide in PB (4°C) until cutting the following day. Brains were cut on a vibratome (Leica, VT1000S, Leica Biosystems, Buffalo Grove, IL, USA) into 40 μm coronal for immunohistochemistry. Sections were stored in 0.1% sodium azide in PB (4°C). Sections from each animal were washed with phosphate-buffered saline, blocked with 1% bovine serum albumin and incubated in primary antibody for GLT-1a (1:1000 dilution in phosphate-buffered saline; gift from J Rothstein’s Laboratory, Johns Hopkins University) overnight at 4 °C. The tissue was then washed in phosphate-buffered saline, and incubated with fluorescent secondary antibody (AlexaFluor488) for 1 h. The intensity of the labeling was quantified using Nikon Imaging software (Nikon Instruments, Melville, NY, USA) in 50 μm intervals from the cellular layer in each region of hippocampus. Electronic images were coded to blind the rater.
RNA extraction, sequencing and analysis
Wet dissected hippocampus from rapidly decapitated rats were flash frozen on dry ice and stored at −80°C. RNA was extracted using the RNAeasy Lipid Kit (QIAGEN Sciences, Germantown, MD, USA) and Qiacube as per the manufacturer’s instructions. Samples were pooled for sequencing and final RNA integrity was checked using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) prior to library preparation. All samples had RNA Integrity Numbers >8. Sequencing libraries were prepared by the Rockefeller University Genomics Core Facility using the TruSeq RNA Library Preparation Kit v2 (Illumina, San Diego, CA, USA) and bar-coded for multiplexing so that all groups could be run in the same flow cell. Single-stranded reads of 100 bp were collected on a HiSeq 2500 (Illumina) at a sequencing depth of ~60 million reads per sample.
Raw data files were uploaded to Galaxy34, 35 and checked for integrity by FastQC. To remove sequencing artifacts and residual adapter sequences, reads were trimmed by 5–10 bp at the 5′ and 3′ ends and then filtered to remove reads with quality scores <20. Reads were aligned to the rat genome (rn5) using TopHat2 (ref. 36 and then loaded into Strand (Agilent) for quantification of read density by DESeq. Differential expression analyses were conducted in Strand using z-tests that were Benjamin–Hochberg corrected for false discovery rates. Venn diagrams and scatter plots based on significant gene lists were generated using Strand (STRand Analysis Software developed at University of California, Davis' Veterinary Genetics Lab, Davis, CA, USA) and Microsoft Excel (Redmond, WA, USA).
Open-array analysis
cDNA was synthesized using the VILO kit (Life Technologies, Carlsbad, CA, USA) using 2 μg of the same RNA submitted for sequencing for each reaction. Open-array plates were loaded from a 384-well plate as described in the standard Open Array protocol (Life Technologies) and run on a Quantstudio 12 k Flex thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). Counts were exported to Microsoft Excel and used to calculate fold change using the ΔΔCt method.37 All values were normalized to pgk1 expression. Open-array fold change values were plotted against RNA-seq results to generate scatter plots and calculate the r2 values (Prism Software, Irvine, CA, USA).
Pathway analysis
Gene lists from RNA-Seq results were uploaded to the DAVID bioinformatics database (http://david.abcc.ncifcrf.gov/home.jsp). The functional clusters, enrichment scores and gene ontology terms for these categories were obtained from the functional annotation clustering tool. In all of the analysis, enrichment scores above 1.3 were considered significant (P<0.05; http://www.nature.com/nprot/journal/v4/n1/full/nprot.2008.211.html).
The enrichment scores from clusters with similar gene ontology terms were used to compare pathways that were altered in both riluzole and control conditions. Histograms were generated in Microsoft Excel.
Comparisons with previous human AD expression studies
AD expression data were analyzed from the GEO and AMP-AD databases and lists of significantly changed genes (P<0.05) were generated for each study. Some studies examined whole hippocampus,38, 39, 40, 41, 42 whereas others used laser capture microdissection to examine specific subregions of hippocampus, such CA1 and CA3 regions43 or CA1 alone,44 or dentate gyrus and entorhinal cortex.45 Therefore, to control for gene differences that arise from using different regions of the hippocampus and identify the most robust findings that were replicated across studies, only genes identified as significant in at least two studies were used for analysis. There were 2024 genes that were upregulated and 1870 genes that were downregulated with AD and met these criteria. These gene lists were analyzed with the DAVID functional annotation clustering tool as described above. The enrichment scores from these clusters were used to compare genes altered with AD to genes altered with riluzole treatment and the pathways with the highest combined enrichment scores are presented.
Genes significantly changed in aging studies in rats (P<0.05) were also analyzed using the GEO database. One study examined gene expression in the CA1 subregion at different points across the life span.46 Gene lists showing differential expression between 3-month- and 23-month-old rats, and between 3-month- and 12-month-old rats were obtained. A second study compared the genes from the whole hippocampus of 4–6-month-40 and 24–26-month-old rats.47 To control for the differences between regions of the hippocampus, only genes that were significant in both studies were used for analysis. There were 508 genes upregulated and 270 genes that were downregulated in aged rats. These gene lists were analyzed with DAVID functional annotation clustering tool as described above. The enrichment scores from these clusters were compared with the enrichment scores of similar clusters obtained from the 443 genes that were downregulated and the 674 genes that were upregulated with age in our RNA-seq data.
Results
Riluzole rescues age-related expression changes in rats
Hippocampal transcriptional profiles change markedly across the life span. In this study, rats between 3 months (young) and 10 months of age (middle-aged) showed 268 genes increased and 254 genes decreased. There were nearly twice as many changes from the 10-month- to 14-month-old (aged) rats, with 674 genes increased and 443 genes decreased, demonstrating that transcriptional changes are stable across adulthood, but accelerate from middle age onwards (Figure 1a; Supplementary Table 1).
Animals treated with the glutamate modulator, riluzole, from 10 until 14 months had 908 genes increased and 927 genes decreased (Figure 1a). Importantly, there is a large overlap of genes (435) that were changed with aging and were also altered by riluzole treatment (Figure 1b). The overlapping genes were plotted to show fold change by age against fold change by riluzole treatment (Figure 1c). The lower right quadrant shows 240 genes that increased with age and were decreased with riluzole treatment. In the upper left quadrant, 96 genes that were decreased with age were increased with riluzole treatment. This profile suggests that riluzole treatment rescues many age-related gene expression changes in the hippocampus.
Differentially expressed genes were organized into functional pathways using the DAVID pathway tools. The pathway classes that were reversed by riluzole are ranked by significance and divided into those that were upregulated with age and downregulated by riluzole (Figure 1d) and pathways that were downregulated with age and upregulated by riluzole (Figure 1e). Many pathways reversed by riluzole treatment were related to synaptic transmission and plasticity. Examples of genes altered by aging that were reversed by riluzole are provided in Table 1. The NMDA receptor subunit NR2b (GRIN2b), a voltage-gated sodium channel subunit (Scn2a1), a calcium/calmodulin protein kinase II alpha (CAMK2A), the microtubule-associated protein 1B (MAP1B), the synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses SHANK3 and the matrix metalloproteinase 9 (MMP9), each decrease with aging and are increased by riluzole treatment, and have been implicated in learning and neuroplasticity.48, 49, 50, 51, 52, 53, 54 In contrast, isoforms of the GABA receptor (GABRA6) are found to increase with aging and are decreased by riluzole treatment and their blockage may improve memory consolidation.50, 55
Notably, several neuroprotective genes were increased with riluzole treatment, including tropomyosin receptor kinase B (TrkB; NTRK2), which is a receptor for brain-derived neurotrophic factor (Supplementary Table2).56 An example of a gene that was significantly decreased with treatment that is implicated in glutamate signaling is the cysteine/glutamate antiporter (xCT;Slc7a11; Supplementary Table 2). xCT exchanges extracellular cysteine for intracellular glutamate which contributes to the regulation of extrasynaptic glutamate levels57 and could be another potential mechanism for the action of riluzole.
Gene pathways implicated in AD are altered by riluzole
Hippocampal data from nine studies characterizing gene expression changes in post-mortem hippocampal tissue from AD patients,38, 39, 40, 41, 42, 43, 44, 45, 87 were obtained from the GEO (Gene Expression Omnibus, NCBI) and AMP-AD (Accelerating Medicines Partnership-Alzheimer’s Disease) databases. Differentially expressed genes identified in at least two of the nine studies were analyzed in DAVID for comparison with the present pathway results (Figure 2a; Supplementary Tables 3). Many of the pathways altered across studies in AD are also changed with riluzole treatment, including ones related to transmission of nerve impulse and synaptic plasticity (Figure 2b). Examples of genes altered in both AD tissue and by riluzole treatment in rats can be found in Table 2. Importantly, the glutamate transporter EAAT2 is significantly decreased in AD, as well as in aging rats, and was rescued by riluzole treatment. Several genes previously implicated in neural transmission and plasticity are diminished in AD and recovered by riluzole treatment, including: ANK3, an integral membrane protein to the underlying spectrin-actin cytoskeleton that mediates synaptic morphology and transmission;58 CAMK2, a calcium/calmodulin kinase protein and major component of the postsynaptic density that is critically involved in induction of synaptic potentiation and memory,51, 59 and Rab3A, a vesicular trafficking protein that is crucial for synaptic plasticity, learning and memory.60, 61
Validation of RNA-seq results
Custom open-array technology allows for high-throughput qRT–PCR analysis of gene expression. Fifty-three genes of interest were selected to assay, given their known roles in neuroplasticity, glutamate signaling, learning and memory. Thirty-nine of these 53 genes were significant between at least one RNA-seq condition (Supplementary Table 5). A strong correlation in fold change was observed between genes identified as significant by RNA-seq and the open-array measurements, with three of the comparisons exhibiting r2 values >0.80 (Figure 3a–d). Conversely, no significant differences in gene expression were observed in the open-array analysis that were not already identified as significant by RNA-seq. This concordance, of both the positive and negative data for these genes, demonstrates that our sequencing analysis reflects reliable changes in gene expression.
To further validate the RNA-seq findings, results were compared against previous reports using microarray technology to study gene expression changes with aging in rat hippocampus.46, 47 Differentially expressed gene lists from rat studies were obtained using the GEO database and analyzed for pathway enrichment using DAVID. Many of the same gene pathways were changed in both the RNA-seq data and previously published reports of aged rats (Supplementary Figure 1). This is despite differences in the exact age of the animals, tissue collection techniques and the different array technologies used across these studies. Together, the bioinformatics and open-array results suggest that the RNA-seq data are representative of age- and riluzole-induced gene expression changes.
Riluzole rescues EAAT2 levels after aging
Riluzole is known to increase EAAT2 expression,22, 26, 27, 28 which helps maintain the correct amount of glutamate in the synaptic cleft.20 Failure of EAAT2 leads to glutamate spillover to the extrasynaptic space, which can cause decreased synaptic efficiency, LTD and excitotoxicity.17, 62 EAAT2 is expressed in neurons, axon terminals and glial cells.63, 64, 65, 66, 67 Previous studies have shown that EAAT2 is decreased with aging and AD.21, 24, 23 RNA-seq results from our rodent experiments confirm that EAAT2 is downregulated with age, but importantly, levels of this gene are rescued by riluzole treatment (Figure 4a). Further, we identified increased immunohistochemical labeling for EAAT2 in the distal portion of CA1 (Figures 4b), which confirms the RNA-seq findings, and suggests a potential mechanism by which riluzole may rescue cognitive function to be further validated in future studies. Importantly, this region corresponds to the area in which increased dendritic spine clustering occurred in response to riluzole treatment.9
Discussion
This study reveals gene expression changes that occur with aging and glutamatergic modulation by riluzole in the rat hippocampus. The majority of transcriptional changes identified occurred from middle-aged to aged rats, rather than from young to middle-aged animals, suggesting that a loss of the transcriptional stability during adulthood occurs with aging (Figure 1a). Importantly, riluzole treatment reversed many of the age-related expression changes in the rat hippocampus (Figures 1b), which primarily occurred in pathways associated with synaptic function (Figures 1d). A similar inverse comparison of the pathways changed by riluzole with those identified in the hippocampus of AD patients demonstrated extensive commonality of affected genes (Figure 2 and Table 2), establishing riluzole’s potential as a therapeutic agent for AD. The expression changes identified by sequencing were highly correlated with qRT–PCR results using open-array technology, which validated over 50 genes of interest (Figure 3). Finally, changes in the levels of EAAT2, a gene known to be increased by riluzole treatment,22, 26, 27 were validated by immunohistochemical labeling in hippocampus (Figure 4). This finding raises the possibility that modulation of glutamate transporters is one mechanism by which riluzole can improve cognitive performance in aging. In addition, these high-throughput studies offer an essential library of new targets that warrant further investigation into their role in glutamatergic transmission in the hippocampus and age-related cognitive decline.
The identification of pathways associated with the maintenance of synaptic health as changed by riluzole (Figures 1d; Table 1) is consistent with previous work that demonstrated riluzole prevented age-related cognitive decline through clustering of dendritic spines,9 an important neuroplastic mechanism that has been shown by electrophysiological studies and computational models to allow non-linear summation of synaptic inputs.11, 68 Some examples of genes implicated in learning and plasticity that are reduced in aging and increased by riluzole treatment include: MMP9, which induces structural spine modifications;54 NR2B, which is important for LTP;31 MAP1B, which helps maintainaince of structural plasticity in the adult brain52 and SHANK3, which has an important role in synaptic regulation.53
Many of riluzole’s effects on the aging rat hippocampus were opposite to the changes observed in human hippocampus from AD patients (Figure 2b). This indicates that many of the key pathways altered by glutamate modulation with riluzole are implicated in the development of AD pathology, suggesting riluzole may have therapeutic potential. Notably, as in aging, the top AD pathways identified also involved synaptic transmission and plasticity and were reversed by riluzole. Some examples of genes recovered by riluzole treatment include: ANK3, which mediates synaptic morphology and transmission;58 CAMK2, involved in induction of synaptic potentiation and memory51, 59 and Rab3A, which is crucial for synaptic plasticity, learning and memory.60, 61 Genes that are consistently altered in AD brains and are reversed by riluzole provide potential future therapeutic targets.
Synaptic dysfunction is a critical pathophysiological mechanism in AD that highly correlates with cognitive decline.69, 70 Amyloid-β (Aβ) and phosphorylated tau toxicities, the hallmarks of the neuropathology of AD, are intimately related to glutamatergic dysregulation, whereby oligomers of Aβ disrupt glutamate uptake, inhibiting LTP through excessive activation of extrasynaptic NMDA receptors.71, 72 Oligomers of Aβ also facilitate LTD71, 73 and decrease surface expression of synaptic NMDA receptors.74 In addition, dysregulated glutamate increases release of Aβ75 and tau76, 77 and enhances tau phosphorylation78 and expression,79 forming a vicious cycle of neurotoxicity. Importantly, previous work has shown that EAAT2 haploinsufficiency accelerates cognitive deficits in an AD mouse model (AβPPswe/PS1ΔE9)80 and EAAT2 overexpression improves cognitive and pathological markers in APPSw,Ind AD mouse model.81 These studies support the hypothesis that improved regulation of glutamatergic signaling via enhanced EAAT2 uptake could potentially mitigate toxicities in AD brains.
Finally, increased immunoreactivity for EAAT2 was observed in the same region as increased spine clustering was previously identified in riluzole-treated rats,9 suggesting a potential mechanism by which riluzole can increase cognitive performance. Glutamate transporters have the key role of regulating synaptic transmission, and thereby learning and memory.20, 25 They prevent glutamate spillover to the extrasynaptic space and minimize cross-talk between neighboring synapses.20, 62 Importantly, they also control the time course of synaptic glutamate.82, 83 More recent work suggests that EAAT2 surface trafficking also shapes synaptic transmission.84 Previous studies have suggested that riluzole increases glutamate uptake through both increased EAAT2 expression and stabilization of the inactivated state of voltage-gated sodium channels.22, 29, 30, 26,28 A recent in vivo study using microelectrode arrays coupled with amperometry has shown that riluzole reduces extrasynaptic glutamate levels and enhances cognitive performance which correlated with the increased glutamate uptake measures.27 These mechanisms of riluzole have been hypothesized to facilitate synaptic glutamatergic activity and to increase glutamate–glutamine cycling while preventing glutamate overflow to the extrasynaptic space.26, 85, 86 Activation of extrasynaptic NMDA receptors has been associated with LTD and excitotoxicity, and it is a likely important mechanism in many neurodegenerative diseases, including AD.17, 18
In conclusion, these findings identify molecular pathways implicated in aging that are rescued by administration of a known glutamate modulator, riluzole. Modeling the expression differences in response to riluzole establishes a framework of changes associated with improved learning and memory, against which other treatments can be compared. Further, many of the pathways changed by riluzole have been implicated across multiple studies in the pathophysiology of AD, suggesting glutamate modulators may represent novel treatments for both age-related cognitive decline and AD. Future studies will seek to conclusively demonstrate whether increased expression and activity of glutamate transporters are the essential mechanism underlying riluzole’s ability to improve cognitive performance.
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Data obtained from the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) Target Discovery Consortium data portal and can be accessed at HYPERLINK. http://dx.doi.org/doi:10.7303/syn2580853; doi:10.7303/syn2580853.
Acknowledgements
This work was supported by DANA Foundation, the Rockefeller University Women & Science Initiative and Alzheimer’s Drug Discovery Foundation to ACP, NIH grant F32 MH102065 to JDG, NIA grant R37 AG06647 to JHM and partial support by grant # UL1 TR000043 from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NCATS).
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Pereira, A., Gray, J., Kogan, J. et al. Age and Alzheimer’s disease gene expression profiles reversed by the glutamate modulator riluzole. Mol Psychiatry 22, 296–305 (2017). https://doi.org/10.1038/mp.2016.33
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DOI: https://doi.org/10.1038/mp.2016.33
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