Hexanucleotide repeat expansions in C9ORF72 underlie a significant fraction of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This study investigates the frequency of C9ORF72 repeat expansions in clinically diagnosed late-onset Alzheimer’s disease (AD).
Design, setting and patients
This case-control study genotyped the C9ORF72 repeat expansion in 872 unrelated familial AD cases and 888 controls recruited as part of the NIA-LOAD cohort, a multi-site collaboration studying 1000 families with two or more individuals clinically diagnosed with late-onset-AD.
Main Outcome Measure
We determined the presence or absence of the C9ORF72 repeat expansion by repeat-primed PCR, the length of the longest non-expanded allele, segregation of the genotype with disease, and clinical features of repeat expansion carriers.
Three families showed large C9ORF72 hexanucleotide repeat expansions. Two additional families carried more than 30 repeats. Segregation with disease could be demonstrated in 3 families. One affected expansion carrier had neuropathology compatible with AD. In the NIA-LOAD series, the C9ORF72 repeat expansions constituted the second most common pathogenic mutation, just behind the PSEN1 A79V mutation, highlighting the heterogeneity of clinical presentations associated with repeat expansions.
C9ORF72 repeat expansions explain a small proportion of patients with a clinical presentation indistinguishable from AD, and highlight the necessity of screening “FTD genes” in clinical AD cases with strong family history.
The apolipoprotein E (APOE) genotype is the major genetic risk factor for Alzheimer's disease (AD). We have access to cerebrospinal fluid (CSF) and plasma APOE protein levels from 641 individuals and genome-wide genotyped data from 570 of these samples. The aim of this study was to test whether CSF or plasma APOE levels could be a useful endophenotype for AD and to identify genetic variants associated with APOE levels. We found that CSF (P = 8.15 × 10−4) but not plasma (P = 0.071) APOE protein levels are significantly associated with CSF Aβ42 levels. We used Mendelian randomization and genetic variants as instrumental variables to confirm that the association of CSF APOE with CSF Aβ42 levels and clinical dementia rating (CDR) is not because of a reverse causation or confounding effect. In addition the association of CSF APOE with Aβ42 levels was independent of the APOE ɛ4 genotype, suggesting that APOE levels in CSF may be a useful endophenotype for AD. We performed a genome-wide association study to identify genetic variants associated with CSF APOE levels: the APOE ɛ4 genotype was the strongest single-genetic factor associated with CSF APOE protein levels (P = 6.9 × 10−13). In aggregate, the Illumina chip single nucleotide polymorphisms explain 72% of the variability in CSF APOE protein levels, whereas the APOE ɛ4 genotype alone explains 8% of the variability. No other genetic variant reached the genome-wide significance threshold, but nine additional variants exhibited a P-value <10−6. Pathway mining analysis indicated that these nine additional loci are involved in lipid metabolism (P = 4.49 × 10−9).
Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-d-galacto-β(1 → 4)-d-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with 15N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-d-galactopyranosyl doublets surrounded by regions of about four or more “naked” mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [61-α(1 → 6)-d-galactosyl-β-d-mannotriaose and 63,64-di-α(1 → 6)-d-galactosyl-β-d-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.
galactose; glycan; lectin; NMR; protein
By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH–π stacking. These issues are resolved by a combination of 15N–1H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH–π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH–π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.
agglutinin; glycolipid; glycoprotein; lectin; sugar code
Apolipoprotein E (APOE) is the most statistically significant genetic risk factor for late-onset Alzheimer’s disease (LOAD). The linkage disequilibrium pattern around the APOE gene has made it difficult to determine whether all of the association signal is derived from APOE or if there is an independent signal from a nearby gene. In this study we attempted to replicate a recently reported association of APOE 3-TOMM40 haplotypes with risk and age at onset.
We used standard techniques to genotype several polymorphisms in the APOE-TOMM40 region in a large case-control series, in a series with cerebrospinal fluid biomarker data and in brain tissue.
We failed to replicate the previously reported association of the polyT polymorphism (rs10524523) with risk and age at onset. We found a significant association between rs10524523 and risk for LOAD among APOE 33 homozygotes but in the opposite direction to the previously reported association (the very-long allele was underrepresented in cases compared to controls in our study (allele frequency: 0.41 vs. 0.48 respectively; p=0.004)). We found no association between rs10524523 and CSF tau or Aβ42 levels or TOMM40 or APOE gene expression.
Although we were not able to replicate the earlier association between the APOE 3-TOMM40 haplotypes and age at onset, we did observe that the polyT polymorphism is associated with risk for LOAD among APOE 33 homozygotes in a large case-control series, but in the opposite direction to the previous report. Additional studies in very large samples will be needed to confirm this association.
A recent genome-wide association study for frontotemporal lobar degeneration with TAR DNA-binding protein inclusions (FTLD-TDP), identified rs1990622 (TMEM106B) as a risk factor for FTLD-TDP. In this study we tested whether rs1990622 is associated with age at onset (AAO) in granulin (GRN) mutation carriers and with plasma GRN levels in mutation carriers and healthy elderly individuals.
Rs1990622 was genotyped in GRN mutation carriers and tested for association with AAO using the Kaplan-Meier and a Cox proportional hazards model.
We analyzed 50 affected and unaffected GRN mutation carriers from four previously reported FTLD-TDP families (HDDD1, FD1, HDDD2 and the Karolinska family). GRN plasma levels were also measured in 73 healthy, elderly individuals.
The risk allele of rs1990622 is associated with a mean decrease of the age at onset of thirteen years (p=9.9×10−7), with lower plasma granulin levels in both healthy older adults (p = 4×10−4) and GRN mutation carriers (p=0.0027). Analysis of the HAPMAP database identified a non-synonymous single nucleotide polymorphism, rs3173615 (T185S) in perfect linkage disequilibrium with rs1990622.
The association of rs1990622 with AAO explains, in part, the wide range in the age at onset of disease among GRN mutation carriers. We hypothesize that rs1990622 or another variant in linkage disequilibrium could act in a manner similar to APOE in Alzheimer’s disease, increasing risk for disease in the general population and modifying AAO in mutation carriers. Our results also suggest that genetic variation in TMEM106B may influence risk for FTLD-TDP by modulating secreted levels of GRN.
Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09×10−5; OR = 2.21; 95%CI = 1.49–3.28) or an unselected population of 12,481 samples (p = 6.82×10−5; OR = 2.19; 95%CI = 1.347–3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS.
We performed whole-exome sequencing in two autopsy-confirmed cases and an elderly unaffected control from a multigenerational family with autosomal dominant neuronal ceroid lipofuscinosis (ANCL). A novel single-nucleotide variation (c.344T>G) in the DNAJC5 gene was identified. Mutational screening in an independent family with autosomal dominant ANCL found an in-frame single codon deletion (c.346_348 delCTC) resulting in a deletion of p.Leu116del. These variants fulfill all genetic criteria for disease-causing mutations: they are found in unrelated families with the same disease, exhibit complete segregation between the mutation and the disease, and are absent in healthy controls. In addition, the associated amino acid substitutions are located in evolutionarily highly conserved residues and are predicted to functionally affect the encoded protein (CSPα). The mutations are located in a cysteine-string domain, which is required for membrane targeting/binding, palmitoylation, and oligomerization of CSPα. We performed a comprehensive in silico analysis of the functional and structural impact of both mutations on CSPα. We found that these mutations dramatically decrease the affinity of CSPα for the membrane. We did not identify any significant effect on palmitoylation status of CSPα. However, a reduction of CSPα membrane affinity may change its palmitoylation and affect proper intracellular sorting. We confirm that CSPα has a strong intrinsic aggregation propensity; however, it is not modified by the mutations. A complementary disease-network analysis suggests a potential interaction with other NCLs genes/pathways. This is the first replication study of the identification of DNAJC5 as the disease-causing gene for autosomal dominant ANCL. The identification of the novel gene in ANCL will allow us to gain a better understanding of the pathological mechanism of ANCLs and constitutes a great advance toward the development of new molecular diagnostic tests and may lead to the development of potential therapies.
The effects of ionizing radiation, with or without the antiangiogenic agent anginex (Ax), on multiple myeloma growth were tested in a SCID-rab mouse model. Mice carrying human multiple myeloma cell-containing pre-implanted bone grafts were treated weekly with various regimens for 8 weeks. Rapid multiple myeloma growth, assessed by bioluminescence intensity (IVIS), human lambda Ig light chain level in serum (ELISA), and the volume of bone grafts (caliper), was observed in untreated mice. Tumor burden in mice receiving combined therapy was reduced to 59% (by caliper), 43% (by ELISA), and 2% (by IVIS) of baseline values after 8 weeks of treatment. Ax or radiation alone slowed but did not stop tumor growth. Four weeks after the withdrawal of the treatments, tumor burden remained minimal in mice given Ax + radiation but increased noticeably in the other three groups. Multiple myeloma suppression by Ax + radiation was accompanied by a marked decrease in the number and activity of osteoclasts in bone grafts assessed by histology. Bone graft integrity was preserved by Ax + radiation but was lost in the other three groups, as assessed by microCT imaging and radiography. These results suggest that radiotherapy, when primed by anti-angiogenic agents, may be a potent therapy for focal multiple myeloma.
The purpose of this study was to delineate the mechanisms by which stromal components of cancer may induce tumour thermotolerance and exploit alterations in stromal and tumour physiology to enhance radiation therapy. The vascular thermoresponse was monitored by daily one-hour 41.5°C heatings in two murine solid tumour models, SCK murine mammary carcinoma and B16F10 melanoma. A transient increase was seen in overall tumour oxygenation for 2–3 days, followed by a progressive decline in tumour pO2 upon continued daily heatings. Vascular thermotolerance was further studied by treating tumours with different heating strategies, i.e. (1) a single 60 min 41.5°C treatment; (2) two consecutive daily treatments of 41.5°C for 60 min; (3) a single 60 min 43°C treatment or (4) two days of 41.5°C for 60 min followed by treatment with 43°C for 60 min on the third day. Pre-heating tumours with mild temperature hyperthermia induced vascular thermotolerance, which was accompanied by evidence of vessel normalisation, i.e. a decrease in microvessel density and an increase in pericyte coverage. Rational scheduling of fractionated radiation during heat-induced increases in tumour oxygen levels rendered a significantly greater, synergistic, tumour growth inhibition. In vitro clonogenic survival responses of the individual cell types associated (endothelial cells, fibroblasts, pericytes and tumour cells) indicated only a direct cellular thermotolerance in endothelial cells. Overall, this suggests that tumour thermotolerance is a physiological phenomenon mediated through improvement of functional vasculature.
Hyperthermia; radiation; thermotolerance; tumour microenvironment; vessel normalisation
Combination of chemotherapeutic agents and angiogenesis inhibitors is now commonly employed in the clinic to treat cancer. Here, we used angiostatic agents anginex and 0118, in combination with the chemotherapeutic irofulven, to treat human ovarian tumor xenografts in mice. General linear mixed models were used to statistically analyze tumor growth curves. Overall, combination of a low, non-toxic dose of irofulven with either angiogenesis inhibitor was more effective at inhibiting tumor growth than any of the single-agent therapies. For example, the anginex/irofulven and 0118/irofulven combinations inhibited tumor growth relative to controls by 92% (p<0.0001) and 96% (p<0.0001), respectively, with the 0118/irofulven combinations yielding 100% complete responses. This study suggests that combination therapy of 0118 or anginex and irofulven may be highly effective in the clinical setting.
anginex; irofulven; galectin-1; angiogenesis; chemotherapy
Recent genome-wide association studies of Alzheimer's disease (AD) have identified variants in BIN1, CLU, CR1 and PICALM that show replicable association with risk for disease. We have thoroughly sampled common variation in these genes, genotyping 355 variants in over 600 individuals for whom measurements of two AD biomarkers, cerebrospinal fluid (CSF) 42 amino acid amyloid beta fragments (Aβ42) and tau phosphorylated at threonine 181 (ptau181), have been obtained. Association analyses were performed to determine whether variants in BIN1, CLU, CR1 or PICALM are associated with changes in the CSF levels of these biomarkers. Despite adequate power to detect effects as small as a 1.05 fold difference, we have failed to detect evidence for association between SNPs in these genes and CSF Aβ42 or ptau181 levels in our sample. Our results suggest that these variants do not affect risk via a mechanism that results in a strong additive effect on CSF levels of Aβ42 or ptau181.
Recent large-scale genetic studies of late-onset Alzheimer’s disease (LOAD) have identified risk variants in CALHM1, GAB2 and SORL1. The mechanisms by which these genes might modulate risk are not definitively known. CALHM1 and SORL1 may alter amyloid-beta (Aβ) levels and GAB2 may influence phosphorylation of the tau protein. In this study we have analyzed disease associated genetic variants in each of these genes for association with cerebrospinal fluid (CSF) Aβ or tau levels in 602 samples from two independent CSF series. We failed to detect association between CSF Aβ42 levels and SNPs in SORL1 despite substantial statistical power to detect association. While we also failed to detect association between variants in GAB2 and CSF tau levels, power to detect this association was limited. Finally, our data suggest that the minor allele of rs2986017, in CALHM1, is marginally associated with CSF Aβ42 levels. This association is consistent with previous reports that this non-synonymous coding substitution results in increased Aβ levels in vitro and provides support for an Aβ-related mechanism for modulating risk for AD.
Alzheimer’s disease; genetics; association; endophenotypes; amyloid; tau; CALHM1; SORL1; GAB2
Alzheimer's Disease (AD) is a complex and multifactorial disease. While large genome-wide association studies have had some success in identifying novel genetic risk factors for AD, case-control studies are less likely to uncover genetic factors that influence progression of disease. An alternative approach to identifying genetic risk for AD is the use of quantitative traits or endophenotypes. The use of endophenotypes has proven to be an effective strategy, implicating genetic risk factors in several diseases, including anemia, osteoporosis and heart disease. In this study we identify a genetic factor associated with the rate of decline in AD patients and present a methodology for identification of other such factors. We have used an established biomarker for AD, cerebrospinal fluid (CSF) tau phosphorylated at threonine 181 (ptau181) levels as an endophenotype for AD, identifying a SNP, rs1868402, in the gene encoding the regulatory sub-unit of protein phosphatase B, associated with CSF ptau181 levels in two independent CSF series . We show no association of rs1868402 with risk for AD or age at onset, but detected a very significant association with rate of progression of disease that is consistent in two independent series . Our analyses suggest that genetic variants associated with CSF ptau181 levels may have a greater impact on rate of progression, while genetic variants such as APOE4, that are associated with CSF Aβ42 levels influence risk and onset but not the rate of progression. Our results also suggest that drugs that inhibit or decrease tau phosphorylation may slow cognitive decline in individuals with very mild dementia or delay the appearance of memory problems in elderly individuals with low CSF Aβ42 levels. Finally, we believe genome-wide association studies of CSF tau/ptau181 levels should identify novel genetic variants which will likely influence rate of progression of AD.
Alzheimer's disease (AD) is the most common neurodegenerative disease affecting more than 4.5 million people in the US. Genetic studies of AD have previously identified pathogenic mutations in three genes (APP, PSEN1 and PSEN2) and polymorphisms in APOE as risk factors. These findings have led to a better understanding of the underlying disease mechanisms. However, half of all AD cases have no known genetic risk factors for disease. Most studies are designed to identify variants associated with risk or age at onset, but rarely cover other important facets of AD, such as disease progression or duration. In this study we have used an established AD biomarker (cerebrospinal fluid tau phosphorylated at threonine 181, ptau181) to find genetic variants that influence levels of ptau181 in the cerebrospinal fluid. This novel and powerful approach has allowed us to identify a genetic factor located in the regulatory subunit of the calcineurin that is also strongly associated with rate of progression of AD. This study is important because it defines a strategy to find novel genetic factors influencing different facets of AD pathobiology including risk, onset and progression.
Low-density lipoprotein receptor-related protein-1 (LRP) on brain capillaries clears amyloid β-peptide (Aβ) from brain. Here, we show that soluble circulating LRP (sLRP) provides key endogenous peripheral ‘sink’ activity for Aβ in humans. Recombinant LRP cluster IV (LRP-IV) bound Aβ in plasma in mice and in Alzheimer’s disease-affected humans with compromised sLRP-mediated Aβ binding, and reduced Aβ-related pathology and dysfunction in a mouse model of Alzheimer mice, suggesting LRP-IV can effectively replace native sLRP and clear Aβ.
Galectins are a sub-family of lectins, defined by their highly conserved β-sandwich structures and ability to bind to β-galactosides, like Gal β1-4 Glc (lactose). Here, we used 15N-1H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of β1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via α1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (Kd) of 10 × 10−6 M, compared to 260 × 10−6 M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the β-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1–carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences.
diffusion; glycan; lectin; NMR spectroscopy; protein Q
Nicotine dependence risk and lung cancer risk are associated with variants in a region of chromosome 15 encompassing genes encoding the nicotinic receptor subunits CHRNA5, CHRNA3 and CHRNB4. To identify potential biological mechanisms that underlie this risk, we tested for cis-acting eQTLs for CHRNA5, CHRNA3 and CHRNB4 in human brain. Using gene expression and disease association studies, we provide evidence that both nicotine-dependence risk and lung cancer risk are influenced by functional variation in CHRNA5. We demonstrated that the risk allele of rs16969968 primarily occurs on the low mRNA expression allele of CHRNA5. The non-risk allele at rs16969968 occurs on both high and low expression alleles tagged by rs588765 within CHRNA5. When the non-risk allele occurs on the background of low mRNA expression of CHRNA5, the risk for nicotine dependence and lung cancer is significantly lower compared to those with the higher mRNA expression. Together, these variants identify three levels of risk associated with CHRNA5. We conclude that there are at least two distinct mechanisms conferring risk for nicotine dependence and lung cancer: altered receptor function caused by a D398N amino acid variant in CHRNA5 (rs16969968) and variability in CHRNA5 mRNA expression.
To test whether a direct antiangiogenic peptide (anginex) and a vascular endothelial growth factor antibody (bevacizumab, Avastin) can transiently normalize vasculature within tumors to improve oxygen delivery, alleviate hypoxia, and increase the effect of radiation therapy.
Tumor oxygenation levels, microvessel density and pericyte coverage were monitored in three different solid tumor models (xenograft human ovarian carcinoma MA148, murine melanoma B16F10, and murine breast carcinoma SCK) in mice. Multiple treatment schedules were tested in these models to assess the influence on the effect of radiation therapy.
In all three tumor models, we found that tumor oxygenation levels, monitored daily in real time, were increased during the first 4 days of treatment with both anginex and bevacizumab. From treatment day 5 onward, tumor oxygenation in treated mice decreased significantly to below that in control mice. This “tumor oxygenation window” occurred in all three tumor models varying in origin and growth rate. Moreover, during the treatment period, tumor microvessel density decreased and pericyte coverage of vessels increased, supporting the idea of vessel normalization. We also found that the transient modulation of tumor physiology caused by either antiangiogenic therapy improved the effect of radiation treatment. Tumor growth delay was enhanced when single dose or fractionated radiotherapy was initiated within the tumor oxygenation window as compared with other treatment schedules.
The results are of immediate translational importance because the clinical benefits of bevacizumab therapy might be increased by more precise treatment scheduling to ensure radiation is given during periods of peak radiosensitivity. The oxygen elevation in tumors by non–growth factor–mediated peptide anginex suggests that vessel normalization might be a general phenomenon of agents directed at disrupting the tumor vasculature by a variety of mechanisms.
Mutations in the progranulin gene (GRN) are causative for Frontotemporal Lobar Degeneration with ubiquitin-immunoreactive neuronal inclusions (FTLD-U). However, additional studies have demonstrated that these variants could be associated with Alzheimer's disease (AD). The influence of GRN genetic variability on susceptibility to AD and on expression levels in a series of neuropathologically-confirmed AD patients as well as in Peripheral Mononuclear Cells (PBMC) and in cells isolated from cerebrospinal fluid (CSF) was investigated. An association study of rs9897526 and rs5848 was carried out in an Italian population and in a replication population of European American patients and controls.
None of the variants tested act as unequivocal susceptibility factor in both populations although a tendency to an increased frequency of rs5848T allele was observed in the Italian group of AD patients. Furthermore, rs9897526 anticipated the onset of the disease in the Italian population. GRN expression in the parietal lobe of AD cases showed a 0.76-fold decrease compared with controls (1.31±0.07 versus 1.73±0.12, P=0.0025). Patients carrying the rs5848 TT genotype had the lowest GRN expression levels (0.96±0.12, P=0.014). Despite no significant differences were found in the relative PBMC and CSF GRN expression in patients compared to controls, stratifying patients according to the presence of rs5848 T allele, a 0.57-fold decrease in GRN mRNA levels over C carriers was found in PBMC (1.22±0.23 versus 0.70±0.12, P=0.04). Similarly to data obtained in brain samples, patients carrying the TT genotype showed the lowest GRN mRNA levels (TT= 0.46±0.14, CC=1.22±0.23; P=0.013). These data argue against a direct role of GRN as a susceptibility factor for sporadic AD but support a role of GRN as a disease-modifying gene, possibly contributing to the failure of neuronal survival.
Alzheimer's Disease (AD); Progranulin (GRN); Single Nucleotide Polymorphism (SNP); Cerebrospinal Fluid (CSF); Peripheral Mononuclear Cells (PBMC)
To identify novel causes of familial neurodegenerative diseases, we extended our previous studies of TAR DNA-binding protein 43 (TDP-43) proteinopathies to investigate TDP-43 as a candidate gene in familial cases of motor neuron disease. Sequencing of the TDP-43 gene led to the identification of a novel missense mutation, Ala-315-Thr, which segregates with all affected members of an autosomal dominant motor neuron disease family. The mutation was not found in 1,505 healthy control subjects. The discovery of a missense mutation in TDP-43 in a family with dominantly inherited motor neuron disease provides evidence of a direct link between altered TDP-43 function and neurodegeneration.
The use of quantitative endophenotypes in genetic studies may provide greater power, allowing for the use of powerful statistical methods and a biological model for the effects of the disease-associated genetic variation. Cerebrospinal fluid (CSF) amyloid beta (Aβ) levels are promising endophenotypes for late-onset Alzheimer’s disease (LOAD) and show correlation with LOAD status and Aβ deposition. In this study, we investigated 29 single nucleotide polymorphisms (SNPs) positive in AlzGene (http://www.alzgene.org) meta-analyses, for association with CSF Aβ levels in 313 individuals. This study design makes it possible to replicate reported LOAD risk alleles while contributing novel information about the mechanism by which they might affect that risk. Alleles in ACE, APOE, BDNF, DAPK1, and TF are significantly associated with CSF Aβ levels. In vitro analysis of the TF SNP showed a change in secreted Aβ consistent with the CSF phenotype and known Alzheimer’s disease variants, demonstrating the utility of this approach in identifying SNPs that influence risk for disease via an Aβ-related mechanism.
Amyloid beta; Alzheimer’s disease; Genetics; Association; Transferrin
gal-1 (galectin-1) mediates cell–cell and cell–extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal β(1→4)-linked galactose side chains. We used 15N–1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8×10−6 M and 40×10−6 M (or 48×10−6 M) respectively, indicating stronger binding than to lactose (Kd=520×10−6 M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-β(1→4)-Gal rather than the traditional Gal-β(1→4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-β(1→4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.
carbohydrate-binding domain; cell surface; galectin–glycan interactions; heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR); NMR diffusion spectroscopy; simple saccharide; CRD, carbohydrate-recognition domain; ERK, extracellular-signal-regulated kinase; gal-1, galectin-1; GRG, galactorhamnogalacturonate glycan [α-(1→2)-l-rhamnosyl-α-(1→4)-D-galacturonosyl]; HSQC, heteronuclear single quantum coherence; Lamp, lysosomal-membrane-associated protein; PFG, pulsed-field gradient
A recent study provisionally identified numerous genetic variants as risk factors for the transition from smoking to the development of nicotine dependence, including an amino acid change in the α5 nicotinic cholinergic receptor (CHRNA5). The purpose of this study is to replicate these findings in an independent dataset and more thoroughly investigate the role of genetic variation in the cluster of physically linked nicotinic receptors, CHRNA5-CHRNA3-CHRNB4, and the risk of smoking.
Individuals from 219 European American families (N=2,284) were genotyped across this gene cluster to test the genetic association with smoking. The frequency of the amino acid variant (rs16969968) was studied in 995 individuals from diverse ethnic populations. In vitro studies were performed to directly test whether the amino acid variant in the CHRNA5 influenced receptor function.
A genetic variant marking an amino acid change showed association with the smoking phenotype (p=0.007). This variant is within a highly conserved region across non-human species, but its frequency varied across human populations (0% in African populations to 37% in European populations). Furthermore, functional studies demonstrated that the risk allele decreased response to a nicotine agonist. A second independent finding was seen at rs578776 (p=0.003), and the functional significance of this association remains unknown.
This study confirms that at least two independent variants in this nicotinic receptor gene cluster contribute to the development of habitual smoking in some populations, and it underscores the importance of multiple genetic variants contributing to the development of common diseases in various populations.
Anginex, a designed peptide 33mer, is known to function both as an antiangiogenic and bactericidal agent. Solving the NMR solution structure of the peptide is key to understand better its structure-activity relationships and to design more bioactive peptides and peptide mimetics. However, structure elucidation of anginex has been elusive due to subunit exchange-induced resonance broadening. Here, we found that performing NMR structural studies in a micellar environment abolishes exchange broadening and allows the structure of anginex to be determined. Anginex folds in an amphipathic, three-stranded antiparallel β-sheet conformation with functionally key hydrophobic residues lying on one face of the β-sheet and positively charged, mostly lysine residues, lying on the opposite face. Structural comparison is made with a homologous, yet relatively inactive peptide, βpep-28. These results contribute to the design of peptidomimetics of anginex for therapeutic use against angiogenically-related diseases like cancer, as well as infectious diseases.
drug design; anti-angiogenic; anti-bacterial; conformation