Fig 2: Reduced CYFIP2 expression does not affect CYFIP1 expression, but leads to increased APP expression at protein but not mRNA level in the hippocampus. ( A ) Representative western blots. ( B ) Quantification showed that CYFIP2 expression is reduced by ~50% in hippocampal synaptosomes of Cyfip2+/- mice (black bar; n = 7) in comparison to wild-type littermates (grey bar, n = 8). CYFIP1 levels are not significantly changed in the same lysates. For quantification CYFIP1 and CYFIP2 expression were normalized to levels of NSE, a neuronal marker. ( C ) Representative western blots. ( D ) Quantification showed that APP protein expression is significantly increased ~2-fold in hippocampal synaptosomes of Cyfip2+/- mice (black bar) in comparison to wild-type littermates (grey bar; n = 8 per genotype). ( E ) Quantitative PCR analysis showed that App mRNA levels are not elevated in hippocampi of Cyfip2+/- mice (black bar) in comparison to wild-type littermates (grey bar) when normalized to the housekeeping gene Hprt ( n = 8 per genotype). Means ± SEM are shown. *** P < 0.001.

Fig 3: Reduced CYFIP2 expression results in phospho-tau accumulation in hippocampal formation and entorhinal cortex of 12-month-old mice.a Representative images of dorsal hippocampal sections from wild-type mice at 3 months and 12 months, and Cyfip2+/- mice at 12 months, immunohistochemically stained for AT-8 (I–III) and PHF-1 (IV–VI) phospho-tau and haemotoxylin to label nuclei. Scale bars are 200 µm. b Analysis of DAB staining in the so and sr of area CA1 and the ml of the dentate gyrus showing increased phospho-tau with normal ageing (area CA3 not shown) (AT-8: CA1 q = 6.57, p < 0.01, CA3 q = 7.26, p < 0.01, DG q = 5.61, p < 0.05; PHF-1: CA1 q = 5.94, p < 0.01, CA3 q = 13.86, p < 0.0001, DG q = 2.19, p = 0.32) and a further increase in 12-month-old Cyfip2+/- mice specifically in the stratum oriens (so) and stratum radiatum (sr) of area CA1 and in the molecular layer (ml) of the dentate gyrus (DG) (AT-8: CA1 q = 7.44, p < 0.01, DG q = 4.4, p < 0.05; PHF-1: CA1 q = 4.77, p < 0.05, DG q = 5.4, p < 0.05). No difference was found in area CA3 (AT-8: q = 0.34, p = 0.97; PHF-1: q = 2.14, p = 0.34, data not shown). c Higher magnification images of hippocampal sections labelled with AT-8 (I–IV) and PHF-1 (V–VIII). d, e Quantification of number of phospho-tau inclusions showing increase in Cyfip2+/- mice (AT-8: q = 5.6, p < 0.05; PHF-1: q = 5.7, p < 0.01), but not with healthy ageing (AT-8: q = 2.7, p = 0.20; PHF-1: q = 3.16, p = 0.12). Inclusions in Cyfip2+/- mice had a characteristic flame shape which were absent in aged wild-types (AT-8: t = 8.17, p < 0.01; PHF-1: t = 5.28, p < 0.05). Scale bars are 10 µm. f Representative images of AT-8 staining in LEC of 12-month-old wild-type and Cyfip2+/- mice (I–II). Higher magnification images showing laminar distribution of AT-8 in the LEC (III–IV). g Quantification of AT-8 positive staining in LEC across all layers showing increase in Cyfip2+/- mice compared with aged wild-types (t = 10.23, p < 0.001). Scale bars are 100 µm. Data represent mean ± SEM and were analysed using one-way ANOVA with Tukey’s correction or Student’s t test. n = 3–4 animals per condition (individual animals shown as white dots). *p < 0.05; **p < 0.01; ***p < 0.001. L2-5 layers 2–5, LD lamina dissecans, LEC lateral entorhinal cortex, ml molecular layer, rf rhinal fissure, so stratum oriens, sr stratum radiatum.

Fig 4: Aß1-42 regulates protein synthesis in neurons via an eIF4E-CYFIP2 axis, resulting in CYFIP2 reduction.a, b Aß1-42 induces increase in eIF4E phosphorylation at Ser209 (q = 4.89, p < 0.05), and an Mnk1 inhibitor (10 µM CGP 57380) reduces this phosphorylation and blocks this effect (q = 0.06, p > 0.99). c,d CPG 57380 treatments prevent Aß1-42-induced protein synthesis in primary neurons (t = 0.014, p = 0.98), as determined by SUnSET. The inhibitor does not alter basal protein synthesis (t = 0.21, p = 0.99, data not shown). e, f Aß1-42 treatment reduces eIF4E binding to CYFIP2, as determined by co-immunoprecipitation (q = 5.73, p < 0.05). This effect is prevented by CGP 57380 treatment (q = 2.01, p = 0.52) which itself does not have an effect (q = 0.66, p = 0.96). g, h Aß1-42 treatment reduces CYFIP2 levels when normalised to levels of neuronal marker NSE (t = 6.92, p < 0.001), which is prevented by CGP 57380 treatment (t = 1.23, p = 0.27). i, j Aß1-42 treatment leads to ubiquitination of CYFIP2 (t = 3.55, p < 0.05), as determined by co-IP, which is prevented by CGP 57380 treatment (t = 0.56, p = 0.60) although the inhibitor alone has no effect (t = 1.03, p = 0.38). Data represent mean ± SEM and were analysed using one-way ANOVA with Tukey’s correction or Student’s t test. n = 3–6 biological replicates (individual experiments shown as white dots). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: Reduced CYFIP2 expression leads to increased BACE1 expression at protein, but not mRNA level, and abnormal amyloid-ß 42 production in the hippocampus. ( A ) Representative western blots. ( B ) Quantification showed that BACE1 protein expression is significantly upregulated by ~40% in hippocampal synaptosomes of Cyfip2+/- mice (black bar) in comparison to wild-type littermates (grey bar; n = 8 per genotype). ( C ) Quantitative PCR analysis showed that Bace1 mRNA levels are not elevated in hippocampus of Cyfip2+/- mice (black bar) in comparison to wild-type littermates (grey bar; n = 8 per genotype). ( D ) An ELISA on whole hippocampi showed significantly elevated amyloid-ß 42 levels by 30% in Cyfip2+/- mutants (black bar; n = 4) versus wild-type littermates (grey bar; n = 5). Means ± SEM are shown. * P < 0.05, ** P < 0.01.