Formin 2 links neuropsychiatric phenotypes at young age to an increased risk for dementia

Abstract Age‐associated memory decline is due to variable combinations of genetic and environmental risk factors. How these risk factors interact to drive disease onset is currently unknown. Here we begin to elucidate the mechanisms by which post‐traumatic stress disorder (PTSD) at a young age contributes to an increased risk to develop dementia at old age. We show that the actin nucleator Formin 2 (Fmn2) is deregulated in PTSD and in Alzheimer's disease (AD) patients. Young mice lacking the Fmn2 gene exhibit PTSD‐like phenotypes and corresponding impairments of synaptic plasticity, while the consolidation of new memories is unaffected. However, Fmn2 mutant mice develop accelerated age‐associated memory decline that is further increased in the presence of additional risk factors and is mechanistically linked to a loss of transcriptional homeostasis. In conclusion, our data present a new approach to explore the connection between AD risk factors across life span and provide mechanistic insight to the processes by which neuropsychiatric diseases at a young age affect the risk for developing dementia.

A Upper panel: Representative images showing NeuN immunoreactivity in the hippocampus of 8-month-old Fmn2 À/À and control mice. Scale bar: 200 lm. Middle panel: Silver staining showing that gross brain morphology is normal in 8-month-old Fmn2 À/À mice. Lower panel: Brain weight is similar in 8-month-old Fmn2 À/À and control mice (n = 5/group). CA1, hippocampal subregion CA1; CA3, hippocampal subregion CA3. B Fmn2 À/À mice show normal explorative behavior as assayed in the open-field test. Upper panel shows a representative image depicting the path in the open field.
Lower panel shows the activity during the 5-min open-field exposure (n = 10/group). C Basal anxiety as measured by the time spent in the four corners (C1-C4) versus the center of the open-field arena. No significant difference was found amongst groups (n = 10/group). D Fmn2 À/À mice show normal motor behavior in the rotarod test (n = 10/group). A FMN2 has been implicated with the regulation of actin dynamics and the actin cytoskeleton is essential for pre-synaptic plasticity and synaptic vesicle dynamics (Hatada et al, 2000;Owe et al, 2009). As such, we hypothesized that alterations to actin-related processes may contribute to the observed phenotypes. The levels of G and Factin were measured in the dentate gyrus isolated from Fmn2 +/+ and Fmn2 À/À mice (n = 4) using the "F-Actin/G-actin In Vivo Assay" from Cytoskeleton, INC. The upper panel shows a representative image of the Western blot performed with the anti-actin antibody provided with the kit. The lower panel shows the quantification of the F/G ratio. There was a significant reduction in F/G-actin ratio in Fmn2 À/À mice (*P = 0.0268, t-test). B We analyzed the genes deregulated in 8-month-old Fmn2 À/À mice for consensus motifs and target transcription factors at promoters of candidate genes using MEME-ChIP platform (Bailey et al, 2009;Machanick & Bailey, 2011) with default parameters. Targets with E-value above 0.05 were considered significant. Promoter regions were defined as AE 2 kb from TSS. We observed a significant enrichment of two motifs; "CCCGCCCC" which is a motif common for SP transcription factor binding sites and "CCGGAAGC" which is a target of ETS/ELK family of transcription factors. C Representative images showing co-localization of ERK1/2 and FMN2 within the hippocampal mossy fiber pathway. Scale bars: 500 lm for low magnification images and 100 lm for high magnification images. mf, mossy fiber; str. luc, stratum lucidum. D Immunoblot analysis showing decreased levels of active ERK1/2, SP1, and ELK1 that was measured using phospho-specific antibodies in the hippocampal dentate gyrus. Right panel shows quantification (n = 5/group; *P < 0.05; t-test). E Immunoblot analysis of hippocampal synaptosomes (Syn) compared to cell homogenate (H), showing that FMN2 and pERK1/2 are present in the pre-synaptic compartment, while pELK1 is absent. Synaptophysin was used to confirm enrichment of synaptosomes (n = 3). F Lysates from hippocampal synaptosomes were used for immunoprecipitation using an antibody detecting pERK1/2 (IP). IgG and homogenate (H) were used as negative and positive controls (n = 3). FMN2 and pERK1/2 but not pELK1 were detected in pERK1/2 immunoprecipitates (IP). These data indicate that FMN2 and pERK1/2 interact at synapses. As expected from our findings showing that pELK1 is not detected at synapses, we could not detect interaction of synaptic pERK1/2 and pELK1. G A model suggesting how synaptic FMN2 may impact on gene expression. FMN2 and ERK1/2 interact at mossy fiber synapses (green lines indicate actin cytoskeleton).
By mechanisms that remain to be determined FMN2 contributes to the activation of ERK1/2 that subsequently activates ELK and SP1 transcription factors which translocate to the nucleus and initiate gene expression. Of course we cannot exclude that FMN2 also affects gene expression by other mechanisms. In the absence of FMN2 (as in Fmn2 À/À mice), the described pathway is less efficient which eventually contributes to altered gene expression. The fact that reduced ERK1/2 signaling is observed in the aging brain and in APP mice may explain why loss of FMN2 accelerates age-associated changes in gene expression.  Figure EV4. Impaired H4K12ac in aged Fmn2 À/À and Fmn2 À/À _APP mice is rescued by Vorinostat treatment.

A Quantification of ChIP-qPCR analysis showing
H4K12ac at the promoter regions of three selected genes that showed decreased expression in 8-month-old Fmn2 À/À mice (compared to wild-type mice) and were increased after Vorinostat treatment. In line with the gene expression data, we observed decreased H4K12ac in 8-month-old Fmn2 À/À mice when compared Fmn2 +/+ animals (one-way ANOVA revealed a significant group difference for Atp5d, P = 0.003; Ndufb7, P = 0.005, and Uqcrq, P = 0.001). Post hoc comparison revealed a significant difference when comparing H4K12ac levels in the placebotreated Fmn2 À/À versus Fmn2 À/À Vorinostattreated or wild-type mice for all three genes (*P < 0.05; **P < 0.01; n = 5/group).

B Quantification of ChIP analysis showing H4K12ac
at the promoter regions of three selected genes that showed decreased expression in 3-monthold Fmn2 À/À _APP mice (compared to 3-monthold APP mice) and were increased after Vorinostat treatment. One-way ANOVA revealed a significant group difference for Cacng6, P = 0.004; Cdh19, P = 0.004, and Htr5b, P = 0.017. Post hoc comparison revealed a significant difference when comparing H4K12ac levels in the placebo-treated APP versus Fmn2 À/À _APP mice and placebo versus Vorinostat-treated Fmn2 À/À _APP mice for all three genes (*P < 0.05; **P < 0.01; n = 5/group). A The total distance traveled during a 5-min open-field exposure was similar amongst groups (n = 7/group). B The time spent in the center or the corners during a 5-min open-field exposure was similar amongst groups (n = 7/group). C The time spent in the open arms (o) and the closed arms (c) of an elevated plus maze was similar amongst groups (n = 9/group). D One-way ANOVA revealed a significant difference in freezing behavior during the memory test (n = 12/group; F = 8.975, P = 0.0006). Post hoc analysis revealed increased freezing behavior when comparing the Fmn2 À/À _APP Vorinostat to the Fmn2 À/À _APP placebo group (*P = 0.139). E Left panel: The escape latency in the water maze test was similar amongst groups (n = 12/group). Right panel: Swim speed was not altered amongst groups. F Mice of the Fmn2 À/À _APP Vorinostat group spent significantly more time in the target quadrant (T) compared to the other quadrants (OQ). Please note that the values for OQ are presented as average of all three other quadrants (*P < 0.03, post hoc analysis, n = 12/group). G The expression of selected genes affected in Fmn2 À/À _APP mice was increased when comparing the Vorinostat group to the placebo group (*P < 0.05; t-test, n = 4/group).

Data information: Error bars indicate SEM.
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