Male-specific epistasis between WWC1 and TLN2 genes is associated with Alzheimer's disease.

Systematic epistasis analyses in multifactorial disorders are an important step to better characterize complex genetic risk structures. We conducted a hypothesis-free sex-stratified genome-wide screening for epistasis contributing to Alzheimer's disease (AD) susceptibility. We identified a statistical epistasis signal between the single nucleotide polymorphisms rs3733980 and rs7175766 that was associated with AD in males (genome-wide significant pBonferroni-corrected=0.0165). This signal pointed toward the genes WW and C2 domain containing 1, aka KIBRA; 5q34 and TLN2 (talin 2; 15q22.2). Gene-based meta-analysis in 3 independent consortium data sets confirmed the identified interaction: the most significant (pmeta-Bonferroni-corrected=9.02*10-3) was for the single nucleotide polymorphism pair rs1477307 and rs4077746. In functional studies, WW and C2 domain containing 1, aka KIBRA and TLN2 coexpressed in the temporal cortex brain tissue of AD subjects (ß=0.17, 95% CI 0.04 to 0.30, p=0.01); modulated Tau toxicity in Drosophila eye experiments; colocalized in brain tissue cells, N2a neuroblastoma, and HeLa cell lines; and coimmunoprecipitated both in brain tissue and HEK293 cells. Our finding points toward new AD-related pathways and provides clues toward novel medical targets for the cure of AD.

Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models.

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development.

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 ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß 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 Aß 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 Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß 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 Aß42 secretion in neuron primary cultures, soluble Aß42 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 Aß production, thereby contributing to Alzheimer's disease development.

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

INTRODUCTION: The 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. RESULTS: We 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. CONCLUSION: Our 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.

Tau pathology modulates Pin1 post-translational modifications and may be relevant as biomarker.

A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.

Pin1 allows for differential Tau dephosphorylation in neuronal cells.

Neurofibrillary degeneration is likely to be related to abnormal Tau phosphorylation and aggregation. Among abnormal Tau phosphorylation sites, pThr231 is of particular interest since it is associated with early stages of Alzheimer's disease and is a binding site of Pin1, a peptidyl-prolyl cis/trans isomerase mainly involved in cell cycle regulation. In the present work, Pin1 level was found strongly increased during neuronal differentiation and tightly correlated with Tau dephosphorylation at Thr231. Likewise, we showed in cellular model that Pin1 allowed for specific Tau dephosphorylation at Thr231, whereas other phosphorylation sites were unchanged. Moreover, cells displaying Tau phosphorylation at Thr231 did not show any Pin1 nuclear depletion. Altogether, these data indicate that Pin1 has key function(s) in neuron and is at least involved in the regulation of Tau phosphorylation at relevant sites. Hence, Pin1 dysfunction, unlikely by nuclear depletion, may have critical consequences on Tau pathological aggregation and neuronal death.

The peptidylprolyl cis/trans-isomerase Pin1 modulates stress-induced dephosphorylation of Tau in neurons. Implication in a pathological mechanism related to Alzheimer disease.

Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress-induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr-231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1-deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate.