In large vertebrate spindles, the majority of microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation in the presence of TPX2, another branching-promoting factor, as HURP's localization to microtubules is enhanced by TPX2 condensation. Lastly, we provide a structure of HURP on the microtubule lattice, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin's structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation.

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARSCoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

SUMMARY The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread.

The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.

Many genetic studies for Alzheimer's disease (AD) have been focused on the identification of common genetic variants associated with AD risk and not on other aspects of the disease, such as age at onset or rate of dementia progression. There are multiple approaches to untangling the genetic architecture of these phenotypes. We hypothesized that the genetic architecture of rate of progression is different than the risk for developing AD dementia. To test this hypothesis, we used longitudinal clinical data from ADNI and the Knight-ADRC at Washington University, and we calculated PRS (polygenic risk score) based on the IGAP study to compare the genetic architecture of AD risk and dementia progression. Dementia progression was measured by the change of Clinical Dementia Rating Sum of Boxes (CDR)-SB per year. Out of the 21 loci for AD risk, no association with the rate of dementia progression was found. The PRS rate was significantly associated with the rate of dementia progression (β= 0.146, p = 0.03). In the case of rare variants, TREM2 (β= 0.309, p = 0.02) was also associated with the rate of dementia progression. TREM2 variant carriers showed a 23% faster rate of dementia compared with non-variant carriers. In conclusion, our results indicate that the recently identified common and rare variants for AD susceptibility have a limited impact on the rate of dementia progression in AD patients.

More than 20 genetic loci have been associated with risk for Alzheimer’s disease (AD), but reported genome-wide significant loci do not account for all the estimated heritability and provide little information about underlying biological mechanisms. Genetic studies using intermediate quantitative traits such as biomarkers, or endophenotypes, benefit from increased statistical power to identify variants that may not pass the stringent multiple test correction in case–control studies. Endophenotypes also contain additional information helpful for identifying variants and genes associated with other aspects of disease, such as rate of progression or onset, and provide context to interpret the results from genome-wide association studies (GWAS). We conducted GWAS of amyloid beta (Aβ42), tau, and phosphorylated tau (ptau181) levels in cerebrospinal fluid (CSF) from 3146 participants across nine studies to identify novel variants associated with AD. Five genome-wide significant loci (two novel) were associated with ptau181, including loci that have also been associated with AD risk or brain-related phenotypes. Two novel loci associated with Aβ42 near GLIS1 on 1p32.3 (β = −0.059, P = 2.08 × 10−8) and within SERPINB1 on 6p25 (β = −0.025, P = 1.72 × 10−8) were also associated with AD risk (GLIS1: OR = 1.105, P = 3.43 × 10−2), disease progression (GLIS1: β = 0.277, P = 1.92 × 10−2), and age at onset (SERPINB1: β = 0.043, P = 4.62 × 10−3). Bioinformatics indicate that the intronic SERPINB1 variant (rs316341) affects expression of SERPINB1 in various tissues, including the hippocampus, suggesting that SERPINB1 influences AD through an Aβ-associated mechanism. Analyses of known AD risk loci suggest CLU and FERMT2 may influence CSF Aβ42 (P = 0.001 and P = 0.009, respectively) and the INPP5D locus may affect ptau181 levels (P = 0.009); larger studies are necessary to verify these results. Together the findings from this study can be used to inform future AD studies.

Genome-wide association studies (GWAS) of disease endophenotypes provide power to identify novel variants and biological information important in disease pathology. Cerebrospinal fluid (CSF) levels of ptau181 and Aβ42, well established AD endophenotypes, allowed us to identify novel loci implicated in AD in a previous GWAS (n=1,296). Potential AD endophenotypes may also help identify novel variants associated with other disease phenotypes. TREM2 has been strongly associated with AD risk, and CSF levels of soluble TREM2 (sTREM2) are a promising AD biomarker. As we previously reported, TREM2 risk variants have highly complex effects on sTREM2 levels. CSF ptau181 and Aβ42 levels were collected from 3,189 unrelated individuals and linear regression was used to determine single nucleotide polymorphisms (SNPs) associated with these CSF proteins. We analyzed independent data sets to determine if associated SNPs were also associated with AD risk, AAO, or disease progression. Bioinformatics analyses were used to determine whether associated loci influenced gene expression. Our previously identified loci (3q28 near GMNC; 9p24.2 within GLIS3) and two novel loci (13q21.1 near PCDH8; 18q23 near NFATC1) were associated with ptau181. Novel variants associated with Aβ42 near GLIS1 (1p32.3) and within SERPINB1 (6p25) were also associated with risk and other AD phenotypes. Bioinformatics analyses revealed the significant variant for Aβ42 also influenced SERPINB1 expression in macrophages and whole blood, and may influence expression in human hippocampus. With a much larger GWAS we identified novel loci associated with CSF levels of ptau181 and with Aβ42 levels. Previous research in AD mouse models reported neutrophil migration across the blood brain barrier toward amyloid plaques. SERPINB1 is a key regulator of neutrophils and our results show it likely influences CSF Aβ42 (representative of plaque load). Our findings suggest adaptive immune response mechanisms and Aβ-associated pathology may be related. We are performing additional analyses to further explore potential impact of these findings in AD. Rare variant, gene-based, and pathway analyses will be used to further explore the role of immune response mechanisms in AD. We will also perform multiple phenotype analyses with CSF sTREM2, tau, ptau181, and Aβ42 to determine potential interactions.

GWAS have been extremely successful in identifying novel loci associated with Alzheimer’s disease (AD), however the search has shifted to look for rare and low frequency variants that will have moderate to strong effects. Since AD has a large familial component, one efficient approach to find those rare genetic variants is to examine families with multiple affected individuals. In this work we will determine if families with LOAD are enriched on rare variants in genes known to be involved on AD, or other neurodegenerative disease (FTD, PD, PSP, ALS, among others), in the largest yet familiar late onset AD (LOAD) dataset, the Familial Alzheimer Sequencing (FASe) project. We have performed whole-exome or whole-genome sequencing on 345 families with 3-5 members each, over a total of 882 cases and 333 nondemented elderly relatives. All samples included in this analysis were recruited by the Knight-ADRC or the NIA-LOAD family study. Families in which the index individual carried a known pathogenic mutation were excluded from the study. Variant discovery was performed following GATK’s best practices. After applying stringent quality controls to variants, genotypes, and samples, we focused our attention on nonsynonymous variants with a minor allele frequency <1% to generate gene-sets to evaluate the enrichment of rare variants in cases over controls, using GSKAT and EPACTS software. We have examined over 256 genes (mendelian and from GWAS studies) known to be involved on AD or other neurodegenerative diseases. We found that 12 genes were nominally significant on the family-based gene-based analyses (G-SKAT). Interestingly, one of those 12 genes was PSEN1(GSKAT P=0.046), in which the association was mainly driven by three missense rare variants (chr14:73614747, p.Pro7Leu; rs199723282, p.Val261Gly; rs200525059, p.Val412Leu). These preliminary results suggest that new risk variants will be identified that will contribute to the understanding of the genetic architecture of Alzheimer’s disease. Analyses are still underway to identify additional variants.

Alzheimer’s disease (AD) pathology appears several years before clinical symptoms, so identifying ways to detect individuals in the preclinical stage is imperative. The cerebrospinal fluid (CSF) Tau/Aβ42 ratio is currently the best known predictor of AD status and cognitive decline, and the ratio of CSF levels of chitinase-3-like 1 protein (CHI3L1, YKL-40) and amyloid beta (Aβ42) were reported as predictive, but individual variability and group overlap inhibits their utility for individual diagnosis making it necessary to find ways to improve sensitivity of these biomarkers.

Genome-wide association studies of 146 plasma protein levels in 818 individuals revealed 56 genome-wide significant associations (28 novel) with 47 analytes. Loci associated with plasma levels of 39 proteins tested have been previously associated with various complex traits such as heart disease, inflammatory bowel disease, Type 2 diabetes and multiple sclerosis. These data suggest that these plasma protein levels may constitute informative endophenotypes for these complex traits. We found three potential pleiotropic genes: ABO for plasma SELE and ACE levels, FUT2 for CA19-9 and CEA plasma levels and APOE for ApoE and CRP levels. We also found multiple independent signals in loci associated with plasma levels of ApoH, CA19-9, FetuinA, IL6r and LPa. Our study highlights the power of biological traits for genetic studies to identify genetic variants influencing clinically relevant traits, potential pleiotropic effects and complex disease associations in the same locus.

Case-control genome-wide association studies (GWAS) have identified loci associated with risk for Alzheimer disease (AD) but they require very large sample sizes and usually identify variants with small effect sizes. GWAS of informative endophenotypes for disease have more power to identify novel variants and provide information about biological mechanisms. Cerebrospinal fluid (CSF) levels of tau, ptau181, and amyloid beta (Aß42) have been well established as endophenotypes for AD. By analyzing data from 1,269 unrelated individuals, we previously identified risk variants for AD that were also associated with CSF levels of tau and ptau181, including a novel variant associated with AD risk. CSF levels of tau, ptau181, and Aß42 were collected from 3,189 unrelated individuals and linear regression was used to determine single nucleotide polymorphisms (SNPs) associated with these CSF proteins. We analyzed independent data sets to determine if associated SNPs were also associated with AD risk, age of symptom onset, or disease progression. We also performed pathway analyses to determine whether SNPs that were suggestive, but did not reach genome-wide significance, can provide information about the biology of AD. We found novel variants associated with ptau181 in OLFM4 (Chromosome 13, p=1.51×10-8) and CTDP1 (Chromosome 18, p=3.05×10-9) loci. In the analyses of CSF levels of Aß42 we found near genome-wide significant signals in GLIS1 (Chromosome 1, p=6.41×10-8) and SERPINB1 (Chromosome 6, p=1.31×10-7) loci. We also replicated our previous findings that variants located in APOE (Chromosome 19, p=1.17×10-31), GLIS3 (Chromosome 9, p=2.63×10-8), and SNAR-I (Chromosome 3, p=2.65×10-10) loci were associated with ptau181 levels. In the previous GWAS we found a novel variant associated with AD risk, tangle pathology, and cognitive decline. Our preliminary analyses of these novel loci for ptau181 and Aß42 levels did not indicate association with AD risk, age at symptom onset, or cognitive decline. By significantly increasing the sample size for our GWAS, we were able to identify novel loci associated with CSF levels of ptau181 and near genome-wide significant associations with Aß42 levels. We are performing additional analyses to determine potential impact of these findings in AD. We will also perform rare variant, gene-based, and additional pathway analyses.

The accumulation of the toxic Aβ peptide in Alzheimer's disease (AD) largely relies upon an efficient recycling of amyloid precursor protein (APP). Recent genetic association studies have described rare variants in SORL1 with putative pathogenic consequences in the recycling of APP. In this work, we examine the presence of rare coding variants in SORL1 in three different European American cohorts: early-onset, late-onset AD (LOAD) and familial LOAD.

ObjectiveAge is the single greatest risk factor for Alzheimer's disease (AD), with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for AD is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid‐beta (Aβ) kinetics in the central nervous system (CNS) of humans.MethodsAβ kinetics were analyzed in 112 participants and compared to the ages of participants and the amount of amyloid deposition.ResultsWe found a highly significant correlation between increasing age and slowed Aβ turnover rates (2.5‐fold longer half‐life over five decades of age). In addition, we found independent effects on Aβ42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aβ42 and a 10‐fold higher Aβ42 reversible exchange rate.InterpretationThese findings reveal a mechanistic link between human aging and the risk of amyloidosis, which may be owing to a dramatic slowing of Aβ turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aβ kinetics associated with aging and amyloidosis suggest opportunities for diagnostic and therapeutic strategies. More generally, this study provides an example of how changes in protein turnover kinetics can be used to detect physiological and pathophysiological changes and may be applicable to other proteinopathies. Ann Neurol 2015;78:439–453

IMPORTANCEThis study assesses factors associated with the most common adverse event following lumbar puncture.OBJECTIVE To identify factors associated with the risk, onset, and persistence of post-dural puncture headache (PDPH). DESIGN, SETTING, AND PARTICIPANTSWe performed univariate and multivariable analyses of 338 lumbar punctures in the Dominantly Inherited Alzheimer Network observational study using linear mixed models, adjusting for participant-level and family-level random effects. MAIN OUTCOMES AND MEASURESWe directly evaluated associations of 3 post-lumbar puncture outcomes (immediate postprocedural headache, PDPH at 24-hour follow-up, and PDPH receiving a therapeutic blood patch) with participant age and sex, positioning, collection method, needle size, needle insertion site, and cerebrospinal fluid (CSF) volume collected. RESULTSThe incidence of adverse events included 73 immediate postprocedural headaches (21.6%), 59 PDPHs at 24-hour follow-up (17.5%), and 15 PDPHs receiving a therapeutic blood patch (4.4%).Greater volume of CSF collected was associated with increased risk of immediate postprocedural headache, largely owing to a nonlinear increase in risk on collection of volumes above 30 mL (odds ratio, 3.73 for >30 mL and 0.98 for <17 mL).In contrast, collection of higher volumes showed a protective effect in decreasing rates of PDPH at 24-hour follow-up and rates of PDPH receiving a therapeutic blood patch (odds ratio, 0.35 per 10 mL).Although differences in needle size did not reach statistical significance, no participant in the 24G needle group received a therapeutic blood patch compared to 8 of 253 for the larger 22G needles.CONCLUSIONS AND RELEVANCE Factors that acutely lower CSF pressure (eg, seated positioning or extracting very high volumes of CSF) may be associated with transient post-lumbar puncture headache, without increasing rates of persistent PDPH or therapeutic blood patch.Collection of up to 30 mL of CSF appears to be well tolerated and safe.

Introduction A recent study found a significant increase of ABCA7 loss-of-function variants in Alzheimer’s disease (AD) cases compared to controls. Some variants were located on noncoding regions, but it was demonstrated that they affect splicing. Here, we try to replicate the association between AD risk and ABCA7 loss-of-function variants at both the single-variant and gene level in a large and well-characterized European American dataset. Methods We genotyped the GWAS common variant and four rare variants previously reported for ABCA7 in 3476 European–Americans. Results We were not able to replicate the association at the single-variant level, likely due to a lower effect size on the European American population which led to limited statistical power. However, we did replicate the association at the gene level; we found a significant enrichment of ABCA7 loss-of-function variants in AD cases compared to controls ( P = 0.0388; odds ratio =1.54). We also confirmed that the association of the loss-of-function variants is independent of the previously reported genome-wide association study signal. Conclusions Although the effect size for the association of ABCA7 loss-of-function variants with AD risk is lower in our study (odds ratio = 1.54) compared to the original report (odds ratio = 2.2), the replication of the findings of the original report provides a stronger foundation for future functional applications. The data indicate that different independent signals that modify risk for complex traits may exist on the same locus. Additionally, our results suggest that replication of rare-variant studies should be performed at the gene level rather than focusing on a single variant.