Gene Expression Profiling of a Hypoxic Seizure Model of Epilepsy Suggests a Role for mTOR and Wnt Signaling in Epileptogenesis

Microarray profiling was used to investigate gene expression in the hypoxic seizure model of acquired epilepsy in the rat, with the aim of characterizing functional pathways which are persistently activated or repressed during epileptogenesis. Hippocampal and cortical tissues were transcriptionally profiled over a one week period following an initial series of seizures induced by mild hypoxia at post-natal day 10 (P10), and the gene expression data was then analyzed with a focus on gene set enrichment analysis, an approach which emphasizes regulation of entire pathways rather than of individual genes. Animals were subjected to one of three conditions: a control with no hypoxia, hypoxic seizures, and hypoxic seizures followed by treatment with the AMPAR antagonist NBQX, a compound currently proposed to be a modulator of epileptogenesis. While temporal gene expression in the control samples was found to be consistent with known processes of neuronal maturation in the rat for the given time window, the hypoxic seizure response was found to be enriched for components of the PI3K/mTOR and Wnt signaling pathways, alongside gene sets representative of glutamatergic, synaptic and axonal processes, perhaps regulated as a downstream consequence of activation of these pathways. Wnt signaling components were also found enriched in the more specifically epileptogenic NBQX-responsive gene set. While activation of the mTOR pathway is consistent with its known role in epileptogenesis and strengthens the case for mTOR or PI3K pathway inhibitors as potential anti-epileptogenic drugs, investigation of the role of Wnt signaling and the effect of appropriate inhibitors might offer a parallel avenue of research toward anti-epileptogenic treatment of epilepsy.


Supporting Figures Legends
(references refer to the main text)

Figure S1
Mitosis gene set under baseline developmental conditions. A. Log2ratios of gene expression relative to the 1h time point, in each tissue separately, are displayed as a heat map, with sampling times post-P10 indicated in hours. Colors saturate for log2-ratio = ±1. Strong repression of many genes is observed, including for the genes coding for the chromosomal passenger proteins aurora kinase B (AURKB) and surviving (BIRC5), and for several centromeric proteins. B. KS plot showing the distribution of log2-ratios at t = 1 week for both tissues. P-value and enrichment are reported for hippocampus only. Hipp = hippocampus, cx = cortex. The arrows point to some of the genes referred to in the main text.

Figure S2
Gene set containing sonic hedgehog (SHH) target genes under baseline developmental conditions. A. Strong repression of most genes in the gene set is observed by t = 1 week. B. Detailed intensity profiles for CXCR4 and TOP2A. C. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure  S1 for key to figure color and scale details.

Figure S3
Neural stem cell markers under baseline developmental conditions. A. Repression of several genes in the gene set is observed by t = 1 week, including CXCR4, CD24 and nestin (NES). B. Detailed intensity profile for CD24. c) KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure S1 for key to figure color and scale details.

Figure S4
Neuronal progenitor markers under baseline developmental conditions. A. Repression of several genes in the gene set is observed by t = 1 week, including doublecortin (DCX) and OLIG2. B. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure S1 for key to figure color and scale details.

Figure S5
A gene set containing multiple neuronal markers under baseline developmental conditions. a) Heat map of gene profiles. b) KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues (P-value and enrichment here displayed for cortex). See Figure S1 for key to figure color and scale details.

Figure S6
A gene set containing an empirical collection of neuronal markers under baseline developmental conditions. The markers were obtained from the Allen Brain Atlas [20]. A. Heat map of gene profiles. B. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure S1 for key to figure color and scale details.

Figure S7
A comprehensive collection of synaptic markers under the baseline developmental conditions. A. Heat map of gene profiles, showing the 22 genes most induced in cortex. Gene descriptions are given on the right, with functional categories color-coded as specified in the legend. B. Detailed intensity profiles for the chloride transporter KCC2. C. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure S1 for key to figure color and scale details.

Figure S8
Astrocytic markers under the baseline developmental conditions. A. Heat map of gene profiles, showing induction of several genes, including connexin 30 (GJB6), glutamine synthesase (GLUL) and GLT-1 (SLC1A2). B. Detailed intensity profiles for GFAP, AQP9 and connexin 30. c) KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. See Figure S1 for key to figure color and scale details.

Figure S9
A gene set containing an empirical collection of oligodendrocytic markers under the baseline developmental conditions. The markers were obtained from the Allen Brain Atlas [20]. A. Heat map of gene profiles, showing very strong induction of many genes, including genes for the myelinating proteins already indicated in Figure 4 (left-hand arrows). A. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues, with very marked leading edges. See Figure S1 for key to figure color and scale details.

Figure S10
NMDA receptor components under the baseline developmental conditions. A. Heat map of gene profiles, showing induction of NR1 and NR2C and repression of NR3A and NR2D. B. Detailed log2-ratio profiles for NR1, NR2C, NR3A and NR2D in hippocampus and C, in cortex. See Figure S1 for key to figure color and scale details.

Figure S11
AMPA receptor components under the baseline developmental conditions. A. Heat map of gene profiles, showing non-significant changes in hippocampus, and repression of GLUR1 and GLUR2 in cortex. B. Detailed log2-ratio profiles for GLUR2 in hippocampus and C, for GLUR1 and GLUR2 in cortex. See Figure S1 for key to figure color and scale details.

Figure S12
GABA receptor components and glutamate decarboxylase 1 and 2 under the baseline developmental conditions. A. Heat map of gene profiles, showing generally non-significant changes. B. Detailed log2-ratio profiles for GABA A -δ (GABRD), GABA A -β1 (GABRB1) and KCC2 in hippocampus and C, in cortex. See Figure S1 for key to figure color and scale details.

Figure S13
Heat map of the differential response to hypoxic seizures for the genes in the In vitro response (BIC or 4AP) group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. Colors represent log2-ratios of intensities of hypoxic seizure samples to that of time-matched controls (red = positive, green = negative values, colors saturate at ±1). Arrows point to genes referred to in the main text.

Figure S14
Heat map of the differential response to hypoxic seizures for the genes in the Hypoxia response group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S15
Heat map of the differential response to hypoxic seizures for the genes in the Glutamatergic/NMDA receptor I group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S16
Heat map of the differential response to hypoxic seizures for the genes in the Glutamatergic/NMDA receptor II group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S17
Heat map of the differential response to hypoxic seizures for the genes in the Synaptic group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S18
Heat map of the differential response to hypoxic seizures for the genes in the Axonal Guidance group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S19
Heat map of the differential response to hypoxic seizures for the genes in the Autism associated group. The group is defined by one of the clusters of enriched gene sets in the membership matrix of Figure 7. See Figure S13 for key to figure color and scale details.

Figure S21
Expression ratios as a function of time for selected IGF-1/PI3K/mTOR pathway genes. The gene name is indicated at the top of each figure. Time at the bottom refers to the duration following hypoxic seizures; the left-hand panels (labeled "hyp") display ratios of hypoxic seizure response to control; the right-hand panels (labeled "hyp + NBQX") displays ratios of response to combined hypoxic seizures and NBQX treatment, to control. The expression ratio R = 1 (no fold change) is indicated by the black horizontal lines. A. IRS1 displays ~ 1.6 fold induction at 12 h. B. AKT3 is very moderately but significantly induced over the time course. C and D. IGF1 and BDNF are not significantly induced.

Figure S22
Heat map of differential expression for the NBQX-responsive genes. The profiles of the 257 genes, each of which is differentially regulated by both hypoxic seizures (HS) relative to control, and by HS+NBQX relative to HS, are shown (these genes form a proper subset of the 1,399 HS-responsive genes displayed in Figure 6). Log2 ratios of gene expression between time-matched samples are shown for the ratio combinations indicated on top. Colors saturate for log2-ratio = ±1.

Figure S23
Expression ratios as a function of time for 11 Wnt genes represented on the microarray. The gene name is indicated at the top of each figure. Time at the bottom refers to the duration following hypoxic seizures; the left-hand panels (labeled "hyp") display ratios of hypoxic seizure response to control; the right-hand panels (labeled "hyp + NBQX") displays ratios of response to combined hypoxic seizures and NBQX treatment, to control. The expression ratio R = 1 (no fold change) is indicated by the black horizontal lines. Only WNT5A and WNT2 are significantly induced.