Targeted gene mutation of E2F1 evokes age-dependent synaptic disruption and behavioral deficits

Abstract

Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often up-regulated and activated in models of neuronal death. However, despite its well-studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In this study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age-dependent olfactory and memory-related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age-dependent loss of post-synaptic protein-95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages, in which behavioral and synaptic perturbations were observed. Finally, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age-dependent behavioral deficits and synaptic perturbations. E2F1 is a transcription factor regulating cell cycle progression and apoptosis. Although E2F1 dysregulation under toxic conditions can lead to neuronal death, little is known about its physiologic activity in the healthy brain. Here, we report significant age-dependent olfactory and memory deficits in mice with dysfunctional E2F1. Coincident with these behavioral changes, we also found age-matched synaptic disruption and persisting reduction in adult neurogenesis. Our study demonstrates that E2F1 contributes to physiologic brain structure and function.

Keywords: E2F; adult neurogenesis; cell cycle; synaptic proteins; transgenic mice.

Figure 1

Behavioral phenotyping of E2f1^tm1 animals. A) Survival curve of E2f1^tm1 mice compared to WT. The median lifespan of E2f1^tm1 mice is approximately 25% less than that of WT (N_WT=27, N_TM=37, p<.01, log-rank and Gehan-Breslow-Wilcoxon tests). B) E2f1^tm1 mice display age-dependent deficits in olfaction. (i–ii.) Odor habituation paradigms using ethyl heptanoate (C9)- ethyl caprylate (C10) ester odorant pairs on (i.) P40 and (iii.) P365+ postnatal age groups (N=7 per group.) Normalized exploratory time is represented as exploratory time in Trial 8/Trial 1. (iii.) Summary of normalized exploratory index of (Trial 8 – Trial 7)/Trial 1 across age groups (N=7 per group). (iv.) General anosmia test on all age groups reveal the similar age-dependent olfactory deficits (N=7 per group.) Black bars represent the WT retrieval time of scented cracker, hatched bars represent that of E2f1^tm1, and gray bars represent that of both genotypes of unscented marble. C) E2f1^tm1 mice display age-dependent deficits in memory. (i–ii.) Short-term 1-hour delay (i.) and long-term 24 hour delay (ii.) novel object recognition and memory task across all age groups (N=7 per group). * denotes statistical significance in the critical comparison made between the normalized exploratory time of object 3 (light gray bars) and that of object 1 (dark gray bars). Bolded text on the x-axis denotes the age groups of when E2f1^tm1 mice failed the task. D) E2f1^tm1 mice display age-dependent elevated anxiety. Light/dark box paradigm (N=7–9 per group) show that E2f1^tm1 (gray bars) spend less time in the light chamber compared to the WT (black bars) at age P90 and greater. Hatched bars at P90 represent results from mice of both genotypes obtained directly from Jackson laboratory and revealed similar results as mice bred in our colony. E) E2f1^tm1 mice have comparable basal activity level as WT. Total beam-break counts in activity chamber of WT (black bars) and E2f1^tm1 (gray bars) on two representative age groups P90 and P365+ (P90 N=5, P365+ N_WT=6 and N_TM=8). F) E2f1^tm1 mice do not exhibit any deficits in motor functioning. (i.–ii.) Latency to fall from the accelerating rotarod of WT (closed symbol) and E2f1^tm1 (open symbol) on two representative age groups P90 (i.) and P365+ (ii.) (P90 N=5, P365+ N_WT=6 and N_TM=8 ). All data are represented as mean ± SEM (*, Student’s t-test; α ≤ 0.05).

Figure 2

E2F1 is predominantly cytoplasmic in vivo and in vitro. A) Coronal hippocampal sections from WT P365+ mice were immunolabeled with E2F1 in green and neuronal dendritic marker MAP2 in red (Top). Higher magnification images of the inset areas reveal E2F1 staining can be punctated where neuritic processes are abundant and the cell bodies are absent (Bottom). B) Primary hippocampal neurons at 14 DIV were labeled with E2F1 in green, MAP2 in red, and the nuclei counterstained with DAPI in blue. Two specific E2F1 antibodies KH95 (Top) and KH20 (Middle) were used and produced similar staining patterns. Condition with no E2F1 primary antibodies was included as negative control (Bottom). C) Exogenous E2F1 expression is predominantly cytoplasmic when overexpressed in primary hippocampal neurons. Primary hippocampal neurons were transfected with E2F1 plasmid at 10DIV, fixed at 14 DIV and labeled with E2F1 in green and MAP2 in red. D) Primary cortical neurons at 21 DIV were fixed and labeled with E2F1 in green, MAP2 in red, and nuclei counterstained with DAPI in blue. Scale bar=30μm.

Figure 3

E2F1 is enriched in the synaptic fractions. A) E2F1 puncta can colocalize with synaptic marker PSD-95. Primary hippocampal neurons at 21 DIV were immunostained with E2F1, PSD-95, and MAP2. Higher magnification of boxed neuritic process is shown as inset. B) E2F1 is enriched in the crude synaptosome isolated from primary hippocampal neuron at 21 DIV. Cell lysates (Lys) were collected using synaptic protein extraction reagent and centrifuged to yield the soluable fraction (Sup) and the synaptosomes (Syn). Enriched synaptic markers PSD-95 and Synapsin were used to validate the isolation of synaptosomes. C) E2F1 is enriched in the synaptoneurosomes isolated from adult mouse cortex and hippocampus. Cortical (Ctx) and hippocampal (Hc) tissues were homogenized (HOM) and the synaptoneurosomes (SN) were isolated. Enriched synaptic markers PSD-95 and vGluT1 were used to validate the isolation of synaptoneurosomes D) E2F1 is enriched in the presynaptic fractions. Pre- and post-synaptic fractions were isolated according to the schematic (Left). Postsynaptic markers PSD-95 was enriched in PSDT1, PSDT2, and PSDT3 whereas presynaptic markers SV2, vGluT1, and synaptophysin were enriched in the SV. All of the synaptic markers were enriched in the crude synaptosomes fraction. Immunoblots for ERK 1/2 are shown in each fractionation experiments as non-synaptic protein loading control. Scale bar=30μm.

Figure 4

Age-dependent synaptic protein perturbations in the E2f1^tm1 animals. A) Immunoblots of various synaptic proteins PSD-95, synGAP, NR1, and NR2A in the brains of WT and E2f1^tm1 mice from two representative age groups P40 and P270. Immunoblots for actin and Coomassie-stained gels are shown as loading controls. B) Quantification of the densitometry analysis of the expression of all tested synaptic proteins displayed as a ratio of the E2f1^tm1 to the WT. There were significant reductions of PSD-95 and synGAP expression in P1, P270, and P365+, of NR1 and NR2A in P270 and P365+, and of GluR2 in P365+ in the E2f1^tm1 mutants compared to the WT. All data are represented as mean ± SEM (*, Student’s t-test; α ≤ 0.05).

Figure 5

Age-dependent reduction in PSD-95 expression in hippocampus and olfactory bulbs. A) Immunoblots of PSD-95 expression in OB (i.) and HC (ii.) across age groups. Coomassie-stained gels and fast green-stained membranes are shown as loading controls. (iii.) Quantification of the densitometry analysis displayed as a ratio of the E2f1^tm1 to the WT. (N=6. *, OB; #, HC) B) (i.) Immunoblots of PSD-95 expression in cerebellum across two representative age groups P90 and P365+. (ii.) Quantification of the densitometry analysis (N=5 P90, N=6 P365+). C) (i.) Representative images of coronal WT and E2f1^tm1 P365+ hippocampal sections immunolabeled with MAP2 (red) and PSD-95 (green) captured at 400x. (ii.) Quantification of the total PSD-95 pixel intensity and the intensity-saturated PSD-95 puncta in the WT and E2f1^tm1 (N=3 for each genotype, 8 sections each, * Student’s t-test; α ≤ 0.05. All data are represented as mean ± SEM.

Figure 6

E2F1 expression increases with age in vitro and in vivo. A) Cytoplasmic and nuclear lysates were collected from primary cortical cultures at various ages in vitro by subcellular fractionation. E2F1 expression elevates in the cytoplasmic fraction but is undetectable in the nuclear fraction. GAPDH serves as a cytoplasmic fraction marker while Lamin A/C serve as a nuclear fraction marker. B) Representative immunoblot of cortical lysates collected from postnatal age 40, 180, 270, 365 for E2F1 expression (left). Immunoblots for GAPDH and Coomassie-stained gels are shown as loading controls. C) Densitometry analysis of E2F1 reveals significant increase starting at P270 and persist until P465 (right, N=5 per group). *denotes p<.05 cmpared to P40, # denotes p<.05 compared to P180, one way-ANOVA Newman-Keuls post-hoc test.

Figure 7

E2f1^tm1 mice display a significant reduction in the number of doublecortin-positive cells in the OB and dentate gyrus of HC. A) Coronal OB and HC sections from WT and E2f1^tm1 P365+ animals immunolabeled with DCX in red. In HC sections, the dentate gyrus is outlined with dashed lines. B) Quantification reveals a strong reduction in the number of newly generated DCX-positive neurons. All data are represented as mean ± SEM. * Student’s t-test; α ≤ 0.05.