Neurodegenerative diseases are the top 3 leading causes of death and are viewed now as systemic diseases. Adaptive immunity including a T-cell response in the central nervous system (CNS) likely contributes to disease pathogenesis. How T cells are primed and recruited to CNS is largely unexplored, due to the complexity of the process and lack of tools and animal models. I will study these questions on the most common genetic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, a GGGGCC repeat expansion in the C9orf72 gene causes haploinsufficiency through reduced C9orf72 protein expression, and gain-of-function toxicities through repeat RNA and its translation to aggregating dipeptide repeats (DPRs). A synergistic role of these pathomechanisms is suspected but not clearly identified. I propose that T cells are the missing piece of the puzzle for the synergistic effects of C9orf72 haploinsufficiency and DRP toxicity and will explore a) whether peripheral antigen-presenting cells (APCs) and CNS microglia present DPRs to prime antigen-specific T-cell response; b) whether C9orf72 haploinsufficiency alters antigen presentation of microglia and APCs; c) whether T cells mediate synergic effects of C9orf72 haploinsufficiency and DRP toxicity. This novel project for the first time addresses peripheral and CNS activation of T cells against DPRs in C9orf72 ALS/FTD and reveals novel mechanism on synergistic effect of C9orf72 haploinsufficiency and DPR toxicity. It offers a unique integration of neurobiological tools, immunological methods, and single-cell-level approaches. It brings solid evidence on the antigen presentation of endogenous aggregating protein to drive antigen-specific T-cell response, which will broad the understanding on ALS/FTD and other neurodegenerative diseases. It presents a promising research trajectory for the identification of new biomarkers, breakthrough therapeutic targets and the development of novel interventions.

Adaptive behaviour to ever-changing environments is crucial for an animals survival in conditions of immediate threat. Meanwhile, there is growing evidence that psychiatric conditions, including anxiety disorders, are at least partly based on maladaptive learning. Neuropeptide signalling plays a key role in regulating synaptic plasticity and hence learning in a state-dependent manner, and dysregulated peptide signalling has been observed in patients suffering from psychiatric disorders. Due to specific expression of peptides and their receptors in distinct cell types and microcircuits, they can contribute to the specificity of neural communication on both temporal and spatial scales. However, the signalling mechanisms and behavioural roles of neuropeptides in individual circuit elements are not well understood. In this proposal, we will use a multimodal approach to determine the role of neuropeptides in innate and learned fear and anxiety states. We will focus on microcircuits of the amygdala, more specifically on distinct interneuron types, which co-release different peptides with their common main transmitter GABA. Using state-of-the-art technology, we will analyse under which conditions neuropeptides are released from interneurons, and probe their causal contribution to fear learning and anxiety states with intersectional genetic interrogations. Large-scale, longitudinal in vivo calcium imaging combined with novel spatial transcriptomics will further reveal peptide impact on circuit computations. As both amygdala circuitry and function as well as neuropeptide expression are evolutionarily conserved, we expect that this proposal will unravel fundamental mechanisms of neural circuit functions for survival. This research will not only provide novel insights into the neural mechanisms of fear and anxiety, but will also hold significant clinical relevance for understanding the underlying pathophysiological processes that contribute to psychiatric disorders.

Liquid-liquid phase separation (LLPS) is a phenomenon inherent to the thermodynamics of liquids, is critical for the development of technologically useful fluids and underlies some of the biggest health changes in our society. LLPS is based on transitions between two different forms of liquid that have the same chemical composition, but distinct energy, entropy and density. Despite the importance of LLPS for technology and health, however, only a very low-resolution view primarily through light microscopy is currently available for LLPS states formed by peptides and proteins. Because of this bottleneck, the interactions, which stabilize liquid droplets, and regulate their biogenesis, as well as a rationale for the biochemical function of LLPS, have remained mysterious. To tackle this massive unmet need, I propose to develop powerful methods of NMR spectroscopy that go far beyond the state-of-the-art and team them up with mechanobiology/force microscopy to break the resolution barrier of polypeptide LLPS and push the description of the internal organization of liquid droplets from micrometer to sub-nanometer. Although highly challenging, the novel methods when successful will (i) disentangle the structure and kinetics of intrinsically disordered proteins within LLPS reaction chambers in space and time, (ii) unravel the nature of chemical reactions in liquid droplets, and (iii) decipher LLPS regulation by posttranslational modifications, nucleic acids and critical changes in cellular environment at atomic resolution. The innovative nature of the proposal is designed to unravel the innermost forces in liquid droplets and to transform our knowledge about the chemistry of liquid phase-separated protein states. Findings from this proposal will provide critical guidance in the development of systems to encapsulate bioactive molecules and to develop better treatments for human diseases.

Liquid-liquid phase separation is a major mechanism for organizing macromolecules, particularly proteins with intrinsically disordered regions, in compartments not limited by a membrane. Many such compartments (also known as biomolecular condensates) have been described, and it is currently agreed that they take over several cellular functions. To do so, they need to interact with other compartments, just as the membrane-bound organelles interact with each other through well-defined contact sites. However, at present no concept exists explaining how membrane-less and membrane-bound organelles interact. I propose here to address this question by determining the molecular mechanisms and functional impact of the interactions between liquid phases and membranes. I hypothesize that a novel type of contact sites between membrane-less organelles and membranes, which I termed dipping contacts, is critical for coupling of diffusion and material properties of condensates to biochemical processes occurring in the membrane-bound compartments. To test this, I will capitalize on a prominent biomolecular condensate that I characterized a few years ago, and which has already been used widely in the literature: the synaptic vesicle (SV) condensate, which clusters SVs together with proteins such as synapsins and synucleins. For this, I have already developed advanced reconstitution tools, single-molecule tracking and genetic code expansion in living neurons, which will enable me to determine: i) how the material properties of SV condensates are regulated, ii) how they recruit specific organelles, while rejecting others, iii) the proteins that mediate signaling and interactions of SV condensates with mitochondria and the ER. Overall, this project will lead to an understanding of the interface between condensates and classical organelles, which is extremely relevant in the context of aggregation-related diseases where faulty inclusions of membranes and proteins play a leading role.

The emerging field of neuroepigenetics investigates processes such as histone-acetylation in the context of neuronal plasticity, memory function and brain diseases. My group has significantly contributed to this novel research field. It is however fair to say that the role of “epigenetics” in memory function is still met with some skepticism in the neurosciences, which is in part due to the fact that many of the current studies have been describing phenomena and mechanistic data to explain how epigenetic processes control memory function in health and disease are comparatively sparse. The major objective of this research proposal is to address this issue and help to consolidate the field of neuroepigenetics by providing insight to the mechanisms by which epigenetic processes contribute to memory formation under physiological and pathological conditions. More specifically I will ask how the epigenetic code is translated into cellular changes that mediate memory formation in health and disease and how can epigenetic mechanisms contribute to the transmission of cognitive phenotypes even across generations. Our results will not only provide import insight to the mechanisms that underlie memory formation but will also lay the basis for the development of novel and improved therapies for age-related cognitive disorders.