Generation and characterization of a mouse with disruption of the <i>Cyfip1</i> gene.

<p>(<b>A</b>) The genomic structure of <i>Cyfip1</i> is shown to scale with larger horizontal boxes representing exons, and the first (ATG) and last (Stop) coding exons indicated. The diagram shows the site of the gene-trap insertion (identified as an LTR-flanked Trapping casette) in intron 1 (5′ to the first coding exon), in order to generate mice with a disruption of the <i>Cyfip1</i> gene. (<b>B</b>) Synaptoneurosome preparations from wildtype (Wt) and <i>Cyfip1</i> heterozygous (Het) mice were subjected to quantitative immunoblotting with an antibody to Cyfip1, with actin as a reference protein. The migration of molecular weight markers is shown on the left (in kDa). (<b>C</b>) Brain mRNA from wildtype (black bars) and <i>Cyfip1</i> heterozygous (white bars) mice were subjected to qPCR for the indicated genes. (<b>D</b>) Quantification of Cyfip1 and Fmrp by immunoblotting of extracts from wildtype (black bars) and heterozygous (white bars) mice. *, P<0.05; **, P = 0.004.</p

DHPG-induced long-term depression is not dependent on protein synthesis or mammalian Target of Rapamycin in <i>Cyfip1</i> heterozygotes.

<p>(<b>A</b>) LTD was induced by DHPG (50 µM for 5 minutes, indicated by the short horizontal bar) in hippocampal slices from wildtype (Wt) and <i>Cyfip1</i> heterozygous (Het) mice. LTD is significantly enhanced in the heterozygotes as compared to wildtype. Inset: Representative EPSP traces recorded before (arrow) or 40 min after DHPG in wildtype and heterozygous animals (scale: 10 ms and 0.5 mV). (<b>B, C</b>) LTD was induced with DHPG in wildtype (B) or <i>Cyfip1</i> heterozygous (C) mice, in the absence (o) or presence (•) of cycloheximide (Cyclohex, 60 µM, indicated by the long horizontal bar). Cycloheximide significantly inhibited LTD in slices from wildtype but not heterozygous animals. (<b>D, E</b>) LTD was induced by DHPG (50 µM, indicated by the short horizontal bar) in hippocampal slices from wildtype (D) or <i>Cyfip1</i> heterozygous (E) mice, in the absence (o) or presence (•) of rapamycin (20 nM, indicated by the long horizontal bar). (<b>F</b>) LTD was induced by DHPG (50 µM, indicated by the short horizontal bar) in hippocampal slices from wildtype (o) or <i>Cyfip1</i> heterozygous (•) mice, the latter in the absence (•) or presence (▪) of both MPEP (10 µM) and LY367385 (indicated by the long horizontal bar). Application of the mGluR1 and mGluR5 antagonists decreased the magnitude of DHPG-induced-LTD in heterozygotes.</p

Normal learning and memory in Morris Water Maze and in fear conditioning but enhanced extinction of inhibitory avoidance in <i>Cyfip1</i> heterozygous mice.

<p>(<b>A</b>) Mice were tested using the Morris Water Maze. Time (s) to travel to the target platform was not significantly different between genotypes. (<b>B</b>) Mice were tested for fear conditioning, with mice receiving shocks at 120 and 180 seconds during training. Testing was performed 24 hours later, in the same test chamber, without footshock. (<b>C</b>) Inhibitory avoidance was measured by latency to enter the dark side of the box associated with prior shock. Extinction of inhibitory avoidance is enhanced in the heterozygotes. The lower panel shows the experimental design. WT, wildtype mice; Het, heterozygous mice; acq, acquisition; ext, extinction; IA, inhibitory avoidance. *, P = 0.027.</p

Basal synaptic properties and long-term potentiation are normal but long-term depression is enhanced in <i>Cyfip1</i> heterozygotes.

<p>(<b>A</b>) Hippocampal slices from 4–6 weeks old wildtype (WT) or Cyfip1 heterozygous (Het) mice were analyzed for baseline synaptic properties, determined by input/output function, representing the relationship between stimulus intensity and the size of the field EPSP slope. (<b>B</b>) Paired-pulse facilitation in the Schaffer collateral-commissural pathway is not different between genotypes over the test interpulse interval of 50 ms. (<b>C</b>) HFS-induced LTP was not significantly different between wildtype (WT) or <i>Cyfip1</i> heterozygous (Het) mice. (<b>D</b>) PP-LFS-induced LTD in <i>Cyfip1</i> heterozygous mice was significantly increased. Inset: Representative EPSP traces recorded before stimulation (arrow) or 60 min after stimulation in wildtype and heterozygous animals (scale: 10 ms and 0.5 mV).</p

Discovery and Replication Case:Control Sample Sets.

<p>Contributing project totals in discovery and replication phases. The totals represent the number of high quality datasets derived from samples.</p

CNVs Enriched in Geriatric Individuals.

<p>CNVs Enriched in Geriatric Individuals.</p

Independent Technology Validation of Presence of CNV Events to Confirm CNVs Detected by Illumina Array.

<p>Error bars denote the standard deviation of quadruplicate runs.</p

Representative Interactions of the Lifespan Longevity Associated Genes Identified.

<p>Gene-gene interactions of independently significant loci. Additional genes implicated by interacting with genes in significantly associated longevity loci. Alternative splicing gene function annotation enrichment of significant loci suggests diverse genetic perturbation with a common biological role. Extension of this functional category to other genes annotated by functional studies with interactions to associated genes implicates potential for screening diverse etiology.</p

Principle Components Analysis of Pediatric and Geriatric Cohorts.

<p>Discovery U.S. Pediatric vs. Icelandic Geriatric A) Principle components (PC) 1 vs. 2 shows distinct clusters likely due to sporadic differential profiles of a specific subset of SNPs between arrays. Since CNV calling is based on multiple neighboring SNPs and differential clustering SNPs are randomly distributed, CNV discovery should not experience significant bias. B) PC2 vs. 3 representing population structure showing some overlap of pediatric and geriatric cohorts C) SNP genotype allele frequency differences genome wide showing close correlation. Replication U.S. Pediatric vs. U.S. Geriatric D) Replication of U.S. pediatric and U.S. geriatric PC1 vs. PC2 showing high overlap unlike panel A U.S. pediatric and Icelandic geriatric E) Geriatric replication cohort in isolation for clarity F) Population structure of pediatric subjects with significantly associated risk CNVs for short lifespan showing broad normal distribution minimizing test statistic inflation for rare variants opposed to tight clustering(37) G) Pediatric replication cohort in isolation for clarity.</p

Manhattan Plot of SNP based CNV Statistics. (A)Deletions and (B)Duplications.

<p>Manhattan Plot of SNP based CNV Statistics. (A)Deletions and (B)Duplications.</p