Max Planck Institute for Biology of Ageing
Stem cells possess a unique metabolic landscape that controls their survival and function. The finetuning of redox and metabolic processes is essential for stem cells’ self-renewal, proliferation, and differentiation, which in turn is fundamental to their regenerative capacity. Deviations from this metabolic balance, by contrast, can contribute to aging and age-associated pathologies, which can particularly impact a person’s quality of life when it is linked to cognitive decline.
It is important to continue deepening our understanding of the aging brain and the potentially rejuvenating neural stem cells (NSCs) that lie dormant or damaged within. The majority of mammalian NSCs remain in a quiescent state and undergo self-renewal via a slow cell cycle. However, certain stimuli activate these quiescent NSCs causing them to proliferate and differentiate into neurons, astrocytes, or oligodendrocytes depending on the received cue. Metabolite utilization via primary metabolic pathways is a key regulator in the context of NSCs’ survival and regenerative function. While transcriptional changes have received scholarly attention for many decades, RNA-binding proteins (RBPs) have only recently taken centre stage as regulators of metabolism. RBPs are a versatile group of proteins that can facilitate short- and long-term metabolic adjustments of cells undergoing cell division and differentiation. Furthermore, RBPs can integrate metabolic stimuli through, for example, post-translational modifications, changes in localization, or metabolite availability.
Our research: Our aim is the development of a combinatorial tool for the metabolism-centric classification of aging NSCs using genetically encoded FRET-based sensors for key metabolites. The powerful RNA interactome-capture method (Perez-Perri et al., 2018) will enable the global identification of proteins differentially associated with RNA in cells with distinct metabolic profiles as measured by our sensors. Candidate proteins with dramatically changed RNA-binding potential will be knocked out, where possible, using CRISPR/Cas9, and the cells will be subjected to transcriptomic and metabolic assessment. The results of this in-depth cellular characterization will be integrated with CLIP datasets of candidate RBPs to understand their role in establishing the metabolic profile of aging NSCs.
Metabolism affects the acetylation and methylation of histones and in turn alters the RNA expression landscape of ageing NSCs. Therefore, it is essential to connect the metabolic state and global RNA expression profiles at the single-cell level. Single-cell transcriptome sequencing will be used on the FRET sensor-expressing cells to characterize RNA expression levels and directly link it to the metabolic state of the cell. This will enable temporal tracing of NSCs while simultaneously assessing metabolic gradients, potentially revealing heterogeneously expressed markers that are relevant for priming cells for a specific age-related fate.
Our goals: Our overarching aim is to utilize the metabolically dynamic system of quiescent and activated NSCs during aging to elucidate the following questions and discover the involvement of canonical and non-canonical RBPs therein:
1. What regulates the metabolic alterations that aging NSCs undergo?
2. What role does metabolism play in the aging process of NSCs?
3. What coordinates cytosolic and mitochondrial metabolic pathways in aging NSCs?
Our successes: One example of a canonical RBP that impacts the metabolic setup of the cell is YBX3. By binding to the 3’ untranslated region, YBX3 stabilizes the transcript of the amino acid transporter SLC7A5 and thereby indirectly alters the availability of large, neutral amino acids in the cell (Cooke et al., 2019). In addition, many essential metabolic enzymes have been identified to bind RNA in different cell types and organisms. Two simplistic modes of RNA–enzyme interactions can be envisioned. On the one hand, metabolic enzymes might moonlight as RBPs and regulate the fate of their target RNAs. On the other hand, RNA might regulate these enzymes, a process we have recently described for the glycolytic enzyme Enolase (Huppertz et al., 2022; Figure). This very large class of non-canonical RBPs might form a novel layer of metabolic regulation.
Our methods/techniques: Our lab strives to find the best tools and methods to answer our questions. This involves the usage of well-established molecular biology-based and biochemical assays to high-throughput screens. It also requires the development of novel techniques to ensure that our research is at the cutting edge and pushes the boundaries of what is currently possible.
Figure 1: The catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells. Taken from Huppertz et al., Molecular Cell, 2022.