Pierre Dourlen

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.

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.

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 Alzheimers 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.

TDP-43 Loss-of-Function Causes Neuronal Loss Due to Defective Steroid Receptor-Mediated Gene Program Switching in Drosophila

TDP-43 proteinopathy is strongly implicated in the pathogenesis of amyotrophic lateral sclerosis and related neurodegenerative disorders. Whether TDP-43 neurotoxicity is caused by a novel toxic gain-of-function mechanism of the aggregates or by a loss of its normal function is unknown. We increased and decreased expression of TDP-43 (dTDP-43) in Drosophila. Although upregulation of dTDP-43 induced neuronal ubiquitin and dTDP-43-positive inclusions, both up- and downregulated dTDP-43 resulted in selective apoptosis of bursicon neurons and highly similar transcriptome alterations at the pupal-adult transition. Gene network analysis and genetic validation showed that both up- and downregulated dTDP-43 directly and dramatically increased the expression of the neuronal microtubule-associated protein Map205, resulting in cytoplasmic accumulations of the ecdysteroid receptor (EcR) and a failure to switch EcR-dependent gene programs from a pupal to adult pattern. We propose that dTDP-43 neurotoxicity is caused by a loss of its normal function.

Drosophila models of tauopathies: what have we learned?

Aggregates of the microtubule-associated protein Tau are neuropathological hallmark lesions in Alzheimers disease (AD) and related primary tauopathies. In addition, Tau is genetically implicated in a number of human neurodegenerative disorders including frontotemporal dementia (FTD) and Parkinsons disease (PD). The exact mechanism by which Tau exerts its neurotoxicity is incompletely understood. Here, we give an overview of how studies using the genetic model organism Drosophila over the past decade have contributed to the molecular understanding of Tau neurotoxicity. We compare the different available readouts for Tau neurotoxicity in flies and review the molecular pathways in which Tau has been implicated. Finally, we emphasize that the integration of genome-wide approaches in human or mice with high-throughput genetic validation in Drosophila is a fruitful approach.

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.

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 Alzheimers 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.

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 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.