Thèse Propagation de Tau de Neurone à Neurone en Fonction de l'Expression de Bin1 dans la Microglie H/F - 59

Établissement : Université de Lille
École doctorale : Biologie Santé de Lille
Laboratoire de recherche : Facteurs de risque et déterminants moléculaires des maladies liées au vieillissement
Direction de la thèse : Jean-Charles LAMBERT ORCID 0000000308297817
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-04-04T23:59:59

Des études d'association pangénomique ont permis de caractériser une partie de la forte composante génétique de la maladie d'Alzheimer (MA), en identifiant 75 loci génétiques associés au risque de MA. L'hypothèse a été émise selon laquelle ces facteurs de risque génétiques devraient être surreprésentés dans les processus physiopathologiques pertinents de la MA. Il est intéressant de noter que la propagation de la protéine tau d'un neurone à l'autre a été proposée comme l'un des processus pathologiques potentiellement dépendants de la génétique. Comme de nombreux facteurs génétiques de la MA s'expriment principalement dans les microglies, nous avons émis l'hypothèse que plusieurs facteurs de risque génétiques sont impliqués dans ce mécanisme par le biais de leur expression microgliale. Le gène BIN1 a particulièrement attiré l'attention en raison de son association avec la pathologie tau. Bien que la protéine tau soit principalement neuronale, des analyses post-génomiques ont indiqué que le gène BIN1 influence la pathologie de la MA par son expression dans les microglies. Afin de comprendre cette contradiction apparente, ce projet utilisera des dispositifs microfluidiques et des co-cultures bidimensionnelles de neurones et de microglies induites par l'homme pour explorer : (i) comment les différentes isoformes, mutations et états de phosphorylation de la protéine tau affectent sa propagation, (ii) le rôle des microglies dans ce processus, et (iii) si le gène BIN1 modifie la propagation de la protéine tau et ses mécanismes lorsqu'il est exprimé dans les neurones ou dans les microglies. En établissant des modèles complets basés sur des cellules humaines afin de commencer à élucider les processus par lesquels la protéine tau se propage, notre projet ouvrira la voie à l'identification de la manière dont les facteurs de risque génétiques tels que BIN1 sont impliqués dans ces processus.

Since the late 2000's, large efforts through genome wide association studies (GWASs) have been invested into the characterization of the genetic component of Alzheimer's disease (AD) (reviewed in (6)), which is estimated to be particularly high for a common, multifactorial disease (60-80% of the attributable risk in twin studies)(7). One of the most reliable pictures of the AD genetic component has been recently resulting in the characterization of 75 loci associated with AD risk and one could hypothesize that there should be an overrepresentation of genetic risk factors in the relevant AD pathophysiological processes. Interestingly, the rate of tau propagation has been proposed as a heritable disease trait in genetically diverse mouse strains (8), suggesting that several genetic risk factors could be involved in this process. Furthermore, it has been found that microglia, in which many AD genetic factors are mainly expressed, are involved in tau spreading (9). Therefore, we can postulate that several genetic risk factors could be involved in this mechanism through their expression in microglia, such as TREM2 (10). However, only a few genes have been tested in this context and among the AD genetic risk factors, BIN1 could be of particular interest. Cumulatively, evidence ranging from clinical/postmortem observations all the way to molecular interactions suggests a strong link between BIN1 and tau pathology in addition to evidence provided by genetics (11). However, Tau is mainly expressed in neurons and astrocytes whereas BIN1 is highly expressed in microglia (the BIN1 sentinel variant has been proposed to specifically modulate its microglia expression). It is thus plausible that microglial BIN1 may be involved in tau trafficking in the AD brain.This project will be organized around three WPs.
WP1. Impact of tau isoforms, mutations and phosphorylation status on tau spreading.
We will first characterize tau characteristics that might favor or protect against tau spreading in our 2D models. For this purpose, we will use the two microfluidic devices described in the preliminary result section to assess spreading from neuron to neuron and we will test different tau constructs overexpression following lentivirus transduction: (i) tau-GFP; (ii) tau-BiFC sensor which allow us to assess tau spreading/aggregation (12); (iii) tau biosensors based on a highly sensitive nanoluciferase binary technology (13) (collaboration with Dr. Cecon, Institut Cohin, Paris) to measure luciferase activity in the pre- or post-synaptic chamber.
Based on these experimental tau models, several tau modifications will be assessed to determine their potential impacts on tau spreading: (i) different tau isoforms. Spreading capacities of 2N3R and 2N4R isoforms will be compared; (ii) pathological mutations responsible for monogenic forms of fronto-temporal dementia. We will further investigate the P301L mutation that we already showed impacting 2N4R tau-GFP spreading (preliminary result section); (iii) Tau phosphorylation status. We will study a mimetic hypophosphorylated 2N4R tau isoform (T181A, S202A, T205A, T212A, S214A, T217A, S235A, S396A, S404A), as well as a 2N4R isoform mutated only at positions T217 and T231 (T217A, T231A). These two epitopes have been chosen for specific analysis as they are particularly relevant for AD diagnosis (14), suggesting an important role in pathophysiological processes, and/or are directly involved in controlling the BIN1-tau interaction (15).
In addition, we will assess potential mechanisms potentially involved in tau spreading by using inhibitors that will be applied in the synaptic chamber of our microfluidic device: (i) heparan to block oligomer spreading through unconventional Non-vesicular Mechanism (16,17); (ii) RAP to block tau monomer spreading through LRP1 uptake (18). Finally, these experiments will be also performed by exposing the synaptic chamber to conditioned medium derived from CHO-APPLDN or CHO-APPwt (19) with normalized concentration of Ab peptides (50nM) with the objective to determine whether tau speading (and the related mechanisms) are dependent on Ab exposures.
WP2. Impact of microglia on tau spreading
For several years, genetics has clearly pointed out the role of microglia in the development of AD and numerous mechanisms have been described to explain its involvement (20). To assess the impact of microglia on tau spreading, we will introduce human-induced microglia into the synaptic chamber at DIV5 before the pre- and postsynaptic neuronal cultures are connected (Fig. 2). Several cell densities will be tested by adding between 500 and 1,000 cells. We will then use live imaging at different times (from DIV7 to DIV28) to monitor tau spreading of our different tau-GFP constructs (or tau-BiFC) from the pre- to post-synaptic chamber, as well as tau location in microglia. Of note, the tau-nanoluciferase system will be only used to quantify tau spreading in the post-synaptic chamber at DIV28 as it requires cell lysate.
At each time point, 5 µl of the culture medium will be taken from the synaptic chamber to quantify the levels of secreted p-tau and total tau. It should be noted that our model does not allow for the quantification of EVs in the synaptic chamber (not enough biological material). As in WP1, heparan or RAP treatments will be performed. In addition, we will determine whether synaptic pruning mechanisms could be involved in tau spreading through complement cascade activation. For this purpose, we will use an anti-C1q antibody which blocks the classical complement cascade in vitro (ANX-M1 antibody)(21). Again, all these experiments will be performed by exposing the synaptic chamber to Ab peptides with the objective of determining whether tau spreading related to microglia activation is dependent on Ab oligomers. In addition, snRNA-seq will be performed by isolating microglia (and some astrocytes that can migrate from the postsynaptic chamber) from the synaptic chamber to help to define potential pathways and key actors related to Ab exposure and tau spreading. If one of the treatments is found to impact tau spreading (heparan, RAP or ANX-M1 antibody) - whether independently or in conjunction with Ab exposure -, snRNA-seq will also be performed to better define the potential mechanisms involved. Although there are now technical options for working with a limited number of cells, pooling of several microfluidic devices will be performed if necessary.
WP3. BIN1 as a modulator of neuron-to-neuron tau spreading.
Following optimal conditions defined in WP1 and WP2, including selection of the most pertinent tau constructs (to limit the number of experiments), we will determine whether BIN1 can influence neuron-to-neuron tau spreading independently or not to microglia. For this purpose, we will use BIN+/- and BIN-/- iPSCs already available in the laboratory (22). This will allow us to modulate BIN1 expression either in neurons in the pre or postsynaptic compartments or in microglia. Difference in tau and p-tau localization as well secretion when BIN1 is underexpressed will be determined and BIN1-tau interaction will be assessed by proximity ligation assay in BIN1wt and BIN1+/- models. As described in WP1 and WP2, the use of inhibitors and antibodies will enable us to evaluate the potential mechanisms of tau spreading that are affected by the under-expression of BIN1, either in neurons or in microglia, following exposure to Ab, or not. As in WP2, snRNA-seq will be performed in the most relevant conditions either in neurons or in microglia to help to define potential pathways and key actors that could be modified as a function of BIN1 under-expression and potentially involved in tau spreading.