Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A novel Alzheimer disease locus located near the gene encoding tau protein

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordAPOE ε4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ε4+ (10 352 cases and 9207 controls) and APOE ε4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ε4 status. Suggestive associations (P<1 × 10-4) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ε4+: 1250 cases and 536 controls; APOE ε4-: 718 cases and 1699 controls). Among APOE ε4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10-9). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ε4+ subjects (CR1 and CLU) or APOE ε4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10-7) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P≤1.3 × 10-8), frontal cortex (P≤1.3 × 10-9) and temporal cortex (P≤1.2 × 10-11). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10-6) and temporal cortex (P=2.6 × 10-6). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ε4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted

Arresting the <i>Drosophila</i> embryo in interphase maintains the ER in an interphase-like state.

<p>(<b>A</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) transgenic embryo injected at metaphase cycle 10 with the DNA replication inhibitor, aphidicolin (APH) and viewed during cycle 11. APH, arrests the embryo in S-phase of cycle 11. In the presence of APH, the ER displayed a loose uniform distribution around the nuclei denoting an interphase-like state. This interphase-like state of the ER persists for greater than 30 minutes without any changes to either localization or structure. This is quantified in the fluorescence intensity traces below (see yellow dotted-lines in merged images). H2-RFP signal inside the nucleus does not increase over this time period as well (arrowheads). (<b>B</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) embryo injected with the protein synthesis inhibitor cycloheximide (CHX) at metaphase of cycle 10 and viewed during interphase of the following cycle. Similar to APH, CHX induced arrest which maintained the ER in an interphase-like state. This is quantified below, as in A. (<b>C</b>) Similar background and approach as A and B. Embryos were injected with an APH+CHX cocktail. ER membrane maintained an interphase-like organization as seen in APH injections alone. Scale bar is 10 μm. Time is in min:sec.</p

3D reconstruction of ER structural changes display a clustering of extended cisternae at the spindle poles during metaphase.

<p>Embryos expressing Pdi-GFP (green) and H2-RFP (red) were fixed and imaged using confocal microscopy. (<b>A</b>) Upper panels (view 1) represent a top view of the nucleus and surrounding ER along the xy-plane and bottom panels (view 2) show the nucleus and ER ~45° -75° tilt along the in the y-plane. Embryos were imaged in the z-direction with a step size of 0.1 μm and subject to 3D reconstruction software. (<b>B</b>) At telophase of cycle 11, the ER is globular and spread along the reforming nuclear envelope and at the midbody (view 1, arrowhead). Exiting mitosis, at interphase, the ER is spread loosely through the cytoplasm outline the nuclear envelope. At prophase, the ER becomes defined and begins to cluster and propagate apically at the spindle poles. These clusters are not uniform in size and appear to be sheet-like structures (view 2, arrows). At metaphase, the clusters are found at the spindle poles and appear to be connected along the spindle area forming a sheath (view 1, arrow). In anaphase, ER cisternal clusters appear with the segregating chromosomes and at the midbody (view 2, arrow). Scale bar is 5 μm.</p

The ER displays dramatic structural and localization changes during mitosis in the early <i>Drosophila</i> embryo.

<p>(<b>A</b>) Mitotic ER dynamics were examined in cycle 11 transgenic <i>Drosophila</i> embryos expressing the ER marker Pdi-GFP and the DNA marker H2-RFP using time-lapse confocal microscopy. Phases of mitosis are listed at the top with relative time (min:sec) listed in the merge panels. During interphase, ER (green) was initially spread loosely around the nucleus. Upon entry into mitosis, ER accumulates around the nucleus and was rapidly converted to thick, perinuclear cisternae upon chromosome condensation (red) and prophase onset. in prometaphase, the ER membrane reorganizes with the developing mitotic spindle and begins to accumulate at the spindle poles. At metaphase and anaphase the ER is aligned with the mitotic spindle and displays movement towards the spindle poles (arrow). During late anaphase and telophase, the ER sees a rapid localization around the segregated, decondensing chromosomes and a localization at the central spindle / midbody (arrowhead). Scale bar is 10 μm. (<b>B</b>) High magnification of mitotic ER changes following a single nucleus used for quantification of ER movements shown in C (asterisk in A). Yellow line denotes fluorescence trace shown in C. Scale bar is 5 μm. (<b>C</b>) Fluorescence intensity trace of ER (green line) and chromosomes (red line) along 20 μm of the developing embryo. ER fluorescence is maximal just adjacent to the nuclear space, but is excluded from the nucleus. During interphase, the ER is evenly distributed throughout the cytoplasm. Intensity around the nucleus increases during mitosis and follows the extension of the spindle. Pdi-GFP signal intensity reached maximum during metaphase (arrows). Condensation and alignment of chromosomes at the metaphase plate are marked by the arrowhead. At telophase, two new nuclear envelopes are formed with a large peak at the remaining midbody.</p

Mitotic Cyc:CDK1 Activity is Necessary for Mitotic ER Dynamics

<p>(<b>A</b>) Cycle 11 transgenic embryo expressing Pdi-GFP / H2-RFP following simultaneous dsRNA-mediated knockdown of Cyclins A,B, and B3. When all three mitotic cyclins were knocked-down, there was a general arrest of the embryo prior to entry into mitosis and a block in ER spatial reorganization events. ER tubules persisted between adjacent nuclei. Chromosomes incompletely condensed (arrowhead) and the ER occasionally invaded the nuclear space (arrow). (<b>B</b>) Quantification of the induced arrest from injection of dsRNA directed at all three mitotic cylins shown in A. Intensities of Pdi-GFP and H2-RFP fluorescence are represented by green and red, respectively. (<b>C</b>) Arrest of ER membrane dynamics was further confirmed by examination of the ER shaping protein, Rtnl1. Injection of dsRNA directed at all three mitotic cyclins into a Rtnl1-GFP / H2-RFP embryo produced an arrest prior to mitotic entry, indicating that Rtnl1 is able to change localization independent of mitotic cyclin/CDK activity. (<b>D</b>) Quantification of arrest seen in C with Rtnl1-GFP in green and H2-RFP in red. Scale bars are 10 μm. Time is in min:sec.</p

Cyclin A is sufficient to drive mitotic ER reorganization events.

<p>(<b>A</b>) Schematic of injection strategy and imaging of <i>Drosophila</i> embryo experiment. Pdi-GFP / H2-RFP transgenic embryos were injected with a mixture of APH and CHX, inducing a cycle 11 interphase arrest. Following this arrest, embryos were injected with an affinity-purified recombinant form of cyclin and observed for changes in ER localization. (<b>B</b>) After injection of GST-CycA, ER (green) gathered near the spindle (yellow arrowhead). Pdi-GFP intensity increases much like WT embryos (arrow). Chromosomes (red) eventually condensed and aligned at the metaphase plate (black arrowhead). The spindle region extended into a fusiform structure, but did not progress beyond this point. There was a lack of ER gathering at spindle poles, as well. (<b>C</b>) Following injection of GST-CycB, embryos remained in an interphase-like state without rearrangement of ER (green) or chromosome (red) condensation. Scale bars are 5 μm. Time is in min:sec.</p

Inhibition of the APC/C maintains the ER in a mitotic state.

<p>(<b>A</b>) Time-lapse confocal images of a cycle 10 Pdi-GFP / H2-RFP transgenic embryo following micro-injection of a dominant-negative form of UbcH10 just prior to entry into mitosis, eventually arresting the embryo in metaphase. The ER displayed normal structural organization and localization changes early in mitosis and relocated to the mitotic spindle upon nuclear envelope breakdown. The embryo then arrested at metaphase and the ER remained along the mitotic spindle. Flares of membrane began to protrude from the perispindle area. ER membrane was steadily lost from the area adjacent to the spindle pole over time (~20 minutes). Yellow trace line indicates plots for B, orange line for C. Scale bar is 5 μm. Time is in min:sec. (<b>B</b>) Fluorescence intensity trace plots of the longitudinal section of a nucleus. The plots are similar to wildtype through metaphase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117859#pone.0117859.g001" target="_blank">Fig. 1C</a>). The later time points show a lack of intense ER at the poles, as seen in wildtype. (<b>C</b>) A plot of fluorescence intensity through a latitudinal section of the spindle highlights the increase in fluorescence around the spindle normally seen during mitosis. At the arrest, multiple peaks are seen where flares of ER expand from the perispindle region (arrows).</p

Mitotic ER rearrangements do not occur until after NEB.

<p>(<b>A</b>) An embryo expressing Rtnl1-GFP and mCh-Tub was observed during cycle 11. Tubulin entered the nuclear space at the 20 second time-point, signaling nuclear envelope breakdown prior to accumulation of ER at the centrosome. At the 40 second time point ER began its rearrangement at the centrosome (arrow). Yellow dotted line denotes fluorescence trace shown in B. Scale bar is 5 μm. (<b>B</b>) Fluorescence intensity traces of Rtnl1 (green line) and mCh-Tub (red line). mCh-Tub displays an intensity peak at 5 μm and 10 μm before NEB (arrowheads). At 20 seconds, mCh-Tub intensity becomes flat indicating NEB, while Rtnl1 intensity begins to form peaks at 5 μm and 10 μm (arrows). Rtnl1 intensity continues to rise and mCh-Tub intensity also rises between 5 μm and 10 μm indicating mitotic spindle formation. Time is in min:sec.</p