5 years (2023 – 2027)

FNR PEARL Programme / University of Luxembourg

Michael T. HENEKA, Sean-Patrick H. RIECHERS, Sofie-Helene SEIBEL, Pablo BOTELLA LUCENA, Dimitri BUDINGER, Vivian Song-Minkyung BAKER, Beatriz ALMEIDA, Zhe LIU (past member), Michele Fabrizio CALIANDRO, Hugo Filipe RANGEL DA COSTA

Dementia has been identified by the Word Health Organization as a major and global health issue. Indeed, we expect an increase from currently 55 million dementia cases to about 150 million in 2050. Approximately two thirds of all dementia patients are suffering from Alzheimer’s disease (AD), a neurodegenerative disorder that leads to memory dysfunction, behavioral disturbances, and loss of all higher cognitive functions.

PEARL MINIALZ aims to investigate direct cellular interactions between microglia and neurons by tunnelling nanotubes (TNTs). Initial data suggest that microglia can establish TNTs with neurons, enabling them to clear pathological protein aggregates and facilitate organelle trafficking between cells. The transport of aggregates via TNTs in AD and its degradation by microglia may play a critical role in preserving neuronal function and integrity. In this project, we will use different disease models to address this question. We will use microglia and neurons differentiated from patient-derived induced pluripotent stem cells (iPSC), as well as primary murine cells and cell lines, to comprehensively investigate, in the context of AD, the formation of TNTs and the functional transfer/modification of cargo through these structures using confocal live imaging techniques. We will further validate findings in fish and rodent models with state-of-the-art 3D super-resolution microscopy and multi-photon in vivo microscopy. Finally, we will validate our findings in microglia and neurons differentiated from iPSCs derived from patients.

In addition we will also assess whether modulating TNT formation between neurons and microglia could open up new therapeutic or diagnostic avenues for the treatment of AD.

4 years (2023 – 2026)

EU Joint Programme – Neurodegenerative Disease Research (JPND)

Hilmi Jaufer THAMEEMUL ANSARI

1. Tarja Malm, University of Eastern Finland, Finland
2. Meaghan O’Reilly, Sunnybrook Research Institute/University of Toronto, Canada
3. Arn M.J.M. van den Maagdenberg, Leiden University Medical Center, Netherlands
4. Denis Vivien, INSERM – Caen Normandie University and Hospital, France
5. Signe Mežinska, University of Latvia, Latvia
6. Else Tolner, Leiden University Medical Centre, Netherlands

The project aims to elucidate key cellular–molecular targets and mechanisms by which the mechanoreceptor PIEZO1 and other mechanoreceptors are used by microglia to sense Aβ deposit stiffness, and to clear Aβ deposits upon cellular activation. We will treat induced pluripotent stem cell (iPSC) differentiated microglia and rodent primary microglia with mechanoreceptor agonists, mechanical stimulation, and stimuli combinations to assess their role in microglial phagocytosis, lysosomal functions, and inflammatory processes in an AD context. We will use CRSIPR-Cas9 constructs or antagonist against respective membrane proteins in iPSC-derived microglia to study loss of function phenotypes.

In murine models of cerebral amyloidosis, we will assess microglial morphology at Aβ deposits and their clearance by microglia using multi-photon in vivo laser scanning microscopy. Results will be validated by FACS-mediated isolation from the adult brain and analysis of microglia containing phagocytosed Aβ. Isolated microglia from brains will also subjected for scRNA-Seq in order to dissect pathways involved in mechanoreceptor-dependent microglial immune activation. Furthermore, we will evaluate neuronal network activity and behavioral phenotypes after activating mechanoreceptors in the models described above. We will monitor neuronal calcium transients and study learning and memory behavior.

In summary, we will generate new information on possible therapeutic effects of microglial mechanoreceptor activation with the aim to maintain microglial homeostasis and clearance capacity.

2 Years (2023 – 2025)

European Molecular Biology Organization (EMBO) Fellowship

Bora TASTAN

Alzheimer’s disease (AD) is characterised by three hallmark pathologies – the accumulation of amyloid-beta (Aβ) in extracellular plaques, the aggregation of hyperphosphorylated tau in intracellular neurofibrillary tangles (NFT), and neuroinflammation.    However, little is currently known about how these pathologies connect. Research over the past decade has shown that microglia, the innate immune cells of the brain, can sense aggregated Aβ. This leads to the assembly of the NLRP3 inflammasome, which consists of the intracellular pattern recognition receptor NLRP3, the adaptor protein ASC, and the effector enzyme caspase-1. Subsequently, microglia produce and secrete several inflammatory mediators, including IL-1β and ASC protein complexes termed as ASC specks. Interestingly, Aβ plaque formation seems to precede tau pathology, and several studies have demonstrated that Aβ fibrils could induce tau seeding and spreading. While microglia depletion, as well as genetic knockout of ASC, were found to reduce spread in an in vivo tau spread model, the influence of the NLRP3 inflammasome on neuronal tau pathology and tau pathology downstream of Aβ has yet to be elucidated. We hypothesize that genetic knockout of either ASC or Nlrp3 not only affects the transcriptome in microglia, but also induces transcriptomic changes in astrocytes and neurons in a non-cell-autonomous manner. We further predict that the NLRP3 inflammasome is important for cellular senescence in tauopathies and for the formation and maintenance of neuronal spines. We will also test for the ability of the NLRP3 inflammasome to modulate Aβ-induced tau pathology and investigate if isolated ASC specks are able to induce tau phosphorylation and tangle formation.

4 years (2022 – 2026)

The Gerhard and Ilse Schick Foundation (Gerhard und Ilse Schick Stiftung)

Masanori ITAKURA, Beatriz ALMEIDA,Adrien RAUH (past member), Archontia Maria ANANIADOU (past member)

AC Immune SA, Switzerland

In Alzheimer’s Disease (AD), evidence show that ASC speck formation downstream of the NLRP3 inflammasome activation underlies the deposition of amyloid-beta (Aβ). ASC specks can be defined as micrometer sized protein complexes with complex physiological roles arising from ASC-dependent inflammasome activation. Once released into the extracellular space in response to pyroptosis, ASC specks were shown to directly interact with Aβ and serve as a seed for Aβ aggregation  in vitro and in vivo. ASC specks are therefore promising targets for novel therapeutic approaches aiming at slowing down AD progression. Our project consists of using diverse molecular biology and imaging techniques to further define the dynamics of pyroptosis and ASC speck release. We will further evaluate if anti-ASC antibodies prevent or slow-down Aβ aggregation induced by ASC specks in vitro and in vivo.

2 years (2023 – 2025)

University of Luxembourg (IAS Audacity)

Laurent MARICHAL, Archontia Maria ANANIADOU (past member)

Theoretical Chemical Physics research group (Prof. Alexandre TKATCHENKO) at the University of Luxembourg

During inflammatory responses, reactive oxygen and nitrogen species are produced and interact promiscuously with endogenous molecules, including aggregation-prone peptides. In the context of amyloid diseases, such as Alzheimer’s disease (AD), it is well-established that inflammatory damage can exacerbate the aggregation and neurotoxicity of amyloid-beta (Aβ) peptides. However, the precise mechanisms underlying this process remain unclear. Notably, under nitro-oxidative stress Aβ can undergo a post-translational modification called nitration, as it was shown in Alzheimer’s disease (AD) patient brains. Furthermore, nitrated Aβ seems to form aggregates at a different rate compared to unmodified Aβ. The exact link between nitrated Aβ and AD is currently not understood. Therefore, the goal of this project is to combine experimental and computational approaches in order unravel the mechanisms behind the effect of Aβ nitration in the context of AD. This project involves a collaboration with the Theoretical Chemical Physics department at UL.

4 years (2023 – 2026)

PI funding (University of Luxembourg)

Shilauni DADWAL

The regulation of the NLRP3 inflammasome, a critical component of the innate immune system, is a complex and dynamic process. In this project, we will delve into how NEK7, MARK4, GBP5, PKR, AKT, BTK, and other proteins regulate the NLRP3 inflammasome, and explore their potential connections in the downstream cascade of ASC-dependent and independent pathways.

NEK7 has been identified as a critical player in NLRP3 inflammasome activation. Indeed, NEK7 binding induces a conformational change in NLRP3, allowing its oligomerization and subsequent inflammasome assembly. MARK4 kinase is also implicated in NLRP3 inflammasome regulation. Guanylate-binding proteins (GBPs) can promote NLRP3 inflammasome assembly and cytokine release during specific infections. Protein kinase PKR has been shown to negatively regulate NLRP3 inflammasome activation by phosphorylating and inhibiting RIPK1 kinase, which is involved in inflammasome signaling. The AKT kinase can phosphorylate NLRP3 and promote its degradation, thus acting as a direct negative regulator. Bruton’s tyrosine kinase (BTK) is associated to NLRP3 inflammasome activation in the context of autoimmune diseases, and BTK inhibitors have shown promise in reducing inflammasome-driven inflammation.

The interconnections between these inflammatory cascade regulators are an exciting area of research. Some of these kinases may act sequentially or synergistically, while others may counteract each other’s effects. Elucidating these interactions will be critical to understand the precise regulatory mechanisms of NLRP3 inflammasome activation in microglia, and consequently develop targeted therapies for inflammatory diseases. Thus, this project aims to provide a comprehensive understanding of how the NLRP3 inflammasome is regulated in canonical, non-canonical, and alternative pathways in rodent and human microglia. By uncovering the intricate interplay between these regulators, we can contribute to a more nuanced understanding of inflammasome biology, which has significant implications for therapeutic interventions in inflammatory diseases.

3-4 years (2023 – 2026/27)

NextImmune2 Doctoral Training Unit (FNR PRIDE Programme)

Shreya Yashraj REGE

Activation of microglia and subsequent formation of NACHT, LRR and PYD domains-containing protein 3 and 1 (NLRP3, NLRP1) have been implicated in aging and neurodegenerative disease. Recent evidence suggests that NLRP3 and NLRP1 have gene regulatory effects that directly connect their assembly to the innate immune metabolism of microglia, presumably through the regulation of key elements of the Krebs cycle. In our project, we will investigate, using human iPSC-derived microglia as an experimental model, how NLRP1-mediated gene regulation influences (i) microglial transcriptomes and (ii) microglial function, which are key for maintaining synaptic integrity and neuronal plasticity. Since microglial senescence has been found to cause tau pathology, a major phenotype of the aging and degenerating brain, we will further study (iii) how NLRP1 affects microglial senescence and renewal in the respective models.

2 Years (2023 – 2025)

PI funding (University of Luxembourg)

Masanori ITAKURA

Amyloid-beta (Aβ), α-synuclein, and TDP-43 are not only known to be etiologic molecules in Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), but have also been shown to undergo several post-translational modifications in response to their surrounding environment. Our project aims to investigate and compare the effects of post-translational modification (nitration, oxidation, glycation, etc.) of pathogenic molecules (Aβ, α-synuclein, TDP-43) on neuroinflammation and neurodegeneration. To achieve this, we will employ artificially modified or isolated molecules from brains of rodent disease models to test a) whether microglia can recognize and internalize these modified proteins, and b) whether and how these modified proteins affect the functionality of microglia (phenotypic change, cytokine production, clearance of aggregated proteins) in vitro and in vivo.

5 years (2022 – 2027)

PI funding (University of Luxembourg)

Arnaud MARY

Recent studies have uncovered neuroinflammation as a new crucial and early player in Alzheimer’s disease (AD), a discovery that has provided us with new potential treatment targets to study and test therapeutically. Pathological neuroinflammation in AD leads to pyroptosis, a pro-inflammatory mode of cell death that is mediated by inflammasomes and gasdermins. While the role of neuroinflammation in the development of AD is well documented, the importance of pyroptosis in this process still needs to be clarified. Hence, our project aims to better understand the mechanisms of microglial pyroptosis in the context of AD, and to test the relevance of inflammasome-dependent pyroptosis inhibition as a new strategy to counteract AD progression.