Julien Chapuis

Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer’s disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14–1.26) (P=3.8 × 10−11)). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Association study of the vascular endothelial growth factor gene with the risk of developing Alzheimer's disease

Numerous observations indicate that cerebrovascular dysfunction contributes to cognitive decline and neurodegeneration in AD. Converging evidence points to a pivotal role for vascular endothelial growth factor (VEGF) in neuronal protection, and the lack of activity of this in neurodegenerative disorders. The VEGF gene is located at 6p21.3, a site several studies have shown to have significant linkage with AD, and a functional polymorphism within the VEGF promoter may alter the risk of developing AD. We assessed the potential impact of this polymorphism on the risk of developing AD in a large French case-control population, and investigated its association with the severity of brain vascular lesions (arteriosclerosis, white matter loss and cerebral amyloid angiopathy) in several brain regions (frontal, temporal, parietal and occipital cortex) in AD. No association of the VEGF promoter polymorphism with the risk of developing AD was observed. No relationship between this polymorphism and vascular pathological changes in AD was detected. Our data indicate that although this polymorphism is functional, it does not confer greater risk for AD, nor modulate the extent of vascular pathology.

Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology

A recent genome-wide association meta-analysis for Alzheimer’s disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of >6000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice. These data indicate that PTK2B acts as an early marker and in vivo modulator of Tau toxicity.

Tau phosphorylation regulates the interaction between BIN1’s SH3 domain and Tau’s proline-rich domain

IntroductionThe application of high-throughput genomic approaches has revealed 24 novel risk loci for Alzheimer’s disease (AD). We recently reported that the bridging integrator 1 (BIN1) risk gene is linked to Tau pathology.ResultsWe used glutathione S-transferase pull-down assays and nuclear magnetic resonance (NMR) experiments to demonstrate that BIN1 and Tau proteins interact directly and then map the interaction between BIN1’s SH3 domain and Tau’s proline-rich domain (PRD) . Our NMR data showed that Tau phosphorylation at Thr231 weakens the SH3-PRD interaction. Using primary neurons, we found that BIN1-Tau complexes partly co-localize with the actin cytoskeleton; however, these complexes were not observed with Thr231-phosphorylated Tau species.ConclusionOur results show that (i) BIN1 and Tau bind through an SH3-PRD interaction and (ii) the interaction is downregulated by phosphorylation of Tau Thr231 (and potentially other residues). Our study sheds new light on regulation of the BIN1/Tau interaction and opens up new avenues for exploring its complex’s role in the pathogenesis of AD.

Genome-wide, high-content siRNA screening identifies the Alzheimer’s genetic risk factor FERMT2 as a major modulator of APP metabolism

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer’s disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the “post-GWAS” era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a β3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aβ peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aβ peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aβ peptide production.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimers disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimers disease development.

Proximity Ligation Assay: A Tool to Study Endogenous Interactions Between Tau and Its Neuronal Partners

Tau is a microtubule associated protein (MAP) that is expressed in neurons of the central nervous system. Tau proteins are deregulated in a group of pathologies, including Alzheimers disease, commonly called tauopathies. Therefore intensive research has been conducted to understand both the regulation of Tau and its involvement in neuronal cellular pathways. Since its originally described interactor tubulin, Tau has been described to interact with several other proteins, including tyrosine kinases (Src, Fyn, Lck) and Phospholipase C-γ. In this chapter, we describe the use of proximity ligation assay as a versatile method to study the endogenous interaction of Tau with these different neuronal partners and use the recently identified Tau interactor Bin1 as case study.

Using High-Throughput Animal or Cell-Based Models to Functionally Characterize GWAS Signals

Purpose of ReviewThe advent of genome-wide association studies (GWASs) constituted a breakthrough in our understanding of the genetic architecture of multifactorial diseases. For Alzheimer’s disease (AD), more than 20 risk loci have been identified. However, we are now facing three new challenges: (i) identifying the functional SNP or SNPs in each locus, (ii) identifying the causal gene(s) in each locus, and (iii) understanding these genes’ contribution to pathogenesis.Recent FindingsTo address these issues and thus functionally characterize GWAS signals, a number of high-throughput strategies have been implemented in cell-based and whole-animal models. Here, we review high-throughput screening, high-content screening, and the use of the Drosophila model (primarily with reference to AD).SummaryWe describe how these strategies have been successfully used to functionally characterize the genes in GWAS-defined risk loci. In the future, these strategies should help to translate GWAS data into knowledge and treatments.

Involvement of IL-33 in Alzheimer's disease

tides (Ab) and biometals in the brain represent a major pathogenic pathway in Alzheimer’s disease (AD) and provide the basis for clinical effective disease-modifying therapy (Duce and Bush, 2010; Lannfelt, 2008). Mapping the micro anatomical distribution of essential and trace elements and isotopes in the brain is essential for understanding AD pathobiology and designing new disease-modifying therapies. To date, mapping the brain metallome has been limited by technical barriers. Here we developed a new technique, High-Resolution Metallomic Imaging Mass Spectrometry (HRMIMS), to perform the first high-resolution multi-elemental and isotopic distribution maps of the brain metallome in the Tg2576 transgenic AD mouse model compared to wild-type control mice. Methods: Mice were procured from the Boston University Alzheimer’s Disease Center Transgenic Mouse Facility. Mouse brains were flash frozen and analyzed by metallomic imaging mass spectrometry (MIMS mapping) using a custom cryogenic cell coupled to nanosecond ultraviolet laser ablation (NULA) and hyphenated high-resolution magnetic sector field ICP-MS at the Boston University Center for Biometals & Metallomics, Boston, MA. Laser wavelength, 213 nm; rate, 5-50 mms-1; spot size: 10 micron (high resolution) to 100 micron (scanning resolution). MIMS analysis was conducted on surround to establish elemental background and calibrated with NIST standards. Results: High resolution metallomic maps generated from Tg2576 (Tg) and wild-type (Wt) mouse brain demonstrated unique elemental and isotopic distribution patterns. Zinc (Zn) brain maps revealed a distinctive distribution pattern marked by cortical lamination, prominent allocortical (hippocampus, amygdala) deposition, and isotopic (67Zn, 70Zn). This pattern was progressively disrupted in Tg mice as a function of age with abnormal Zn accumulation co-localizing with amyloid plaque and Timm’s staining. Simultaneous metallomic maps of the same Wt and Tg brains revealed distinctive elemental and isotopic distribution patterns for other important biometals, including copper (Cu), iron (Fe), selenium (Se), molybdenum (Mo), manganese (Mn), and others. Conclusions: We deployed HR-MIMS analysis to generate detailed quantitative high-resolution spatial distribution maps of essential and trace elements and isotopes in Tg2576 transgenic and wild-type mouse brain at 10-100 micron spatial resolution. This study strongly supports a role for zinc in AD-linked brain pathology.

P1-269: Putative functional urea cycle in the brain of patients with Alzheimer’s disease and association of the ornithine transcarbamylase gene with the risk of developing Alzheimer’s disease

4 in biparietal AD. Methods: Thirty-nine patients with sporadic AD underwent clinical assessment and investigation, including MRI. Neuropsychological testing was performed using graded-difficulty tests of verbal and visual recognition memory and parietal lobe function. Patients scoring 5th percentile on dominant parietal lobe testing, 5th percentile on at least one test of non-dominant parietal lobe function, and 10th percentile on the combined verbal and visual memory tests were defined as biparietal AD; others were classified as typical AD. APOE genotype was determined using standard techniques. We compared the frequency of APOE 4 positive genotype, using Fisher’s exact test and clinical characteristics between groups using unpaired t tests. Results: Ten patients fulfilled criteria for biparietal AD; seven had posterior cortical atrophy on MRI. There were no gender, handedness or disease duration differences between the groups. Biparietal patients were younger at symptom onset (56.1 4.1 vs 65.6 6.9; P 0.001), scored higher on the MMSE (23.1 2.8 vs 19.2 4.3; P 0.01) and were less likely to be APOE 4-positive (2/10 vs 25/29, P .0001). Four biparietal and 29 patients with typical AD had serial volumetric imaging performed one year apart. We found some evidence to suggest that biparietal patients had excess rates of whole brain atrophy compared to patients with typical disease. Conclusions: Despite having younger age at onset, patients with biparietal AD are less likely to be APOE 4positive. We suggest that in these patients, at least in part mediated by lack of APOE 4, the pathological process is directed away from medial temporal structures and toward the parietal lobes. If replicated in larger studies, this finding may have implications for our understanding of the pathogenesis of AD and factors influencing the regional predilection of this and other neurodegenerative diseases.