Induced pluripotent stem cells (iPSCs) have revolutionized the world of regenerative medicine; nevertheless, the exact molecular mechanisms underlying their generation and differentiation remain elusive. Here, we investigated the role of the cell fate determinant TRIM32 in modulating such processes. TRIM32 is essential for the induction of neuronal differentiation of neural stem cells by poly-ubiquitinating cMyc to target it for degradation resulting in inhibition of cell proliferation. To elucidate the role of TRIM32 in regulating somatic cell reprogramming we analysed the capacity of TRIM32-knock-out mouse embryonic fibroblasts (MEFs) in generating iPSC colonies. TRIM32 knock-out MEFs produced a higher number of iPSC colonies indicating a role for TRIM32 in inhibiting this cellular transition. Further characterization of the generated iPSCs indicated that the TRIM32 knock-out iPSCs show perturbed differentiation kinetics. Additionally, mathematical modelling of global gene expression data revealed that during differentiation an Oct4 centred network in the wild-type cells is replaced by an E2F1 centred network in the TRIM32 deficient cells. We show here that this might be caused by a TRIM32-dependent downregulation of Oct4. In summary, the data presented here reveal that TRIM32 directly regulates at least two of the four Yamanaka Factors (cMyc and Oct4), to modulate cell fate transitions.

The balance between stem cell maintenance and differentiation has been proposed to depend on antagonizing ubiquitination and deubiquitination reactions of key stem cell transcription factors (SCTFs) mediated by pairs of E3 ubiquitin ligases and deubiquitinating enzymes. Accordingly, increased ubiquitination results in proteasomal degradation of the SCTF, thereby inducing cellular differentiation, whereas increased deubiquitination stabilizes the SCTF, leading to maintenance of the stem cell fate. In neural stem cells, one of the key SCTFs is c-Myc. Previously, it has been shown that c-Myc is ubiquitinated by the E3 ligase TRIM32, thereby targeting c-Myc for proteasomal degradation and inducing neuronal differentiation. Accordingly, TRIM32 becomes upregulated during adult neurogenesis. This upregulation is accompanied by subcellular translocation of TRIM32 from the cytoplasm of neuroblasts to the nucleus of neurons. However, we observed that a subpopulation of proliferative type C cells already contains nuclear TRIM32. As these cells do not undergo neuronal differentiation, despite containing TRIM32 in the nucleus, where it can ubiquitinate c-Myc, we hypothesize that antagonizing factors, specifically deubiquitinating enzymes, are present in these particular cells. Here we show that TRIM32 associates with the deubiquitination enzyme USP7, which previously has been implicated in neural stem cell maintenance. USP7 and TRIM32 were found to exhibit a dynamic and partially overlapping expression pattern during neuronal differentiation both in vitro and in vivo. Most importantly, we are able to demonstrate that USP7 deubiquitinates and thereby stabilizes c-Myc and that this function is required to maintain neural stem cell fate. Accordingly, we propose the balanced ubiquitination and deubiquitination of c-Myc by TRIM32 and USP7 as a novel mechanism for stem cell fate determination.

The vast majority of Parkinson's disease cases are idiopathic. Unclear etiology and multifactorial nature complicate the comprehension of disease pathogenesis. Identification of early transcriptomic and metabolic alterations consistent across different idiopathic Parkinson's disease (IPD) patients might reveal the potential basis of increased dopaminergic neuron vulnerability and primary disease mechanisms. In this study, we combine systems biology and data integration approaches to identify differences in transcriptomic and metabolic signatures between IPD patient and healthy individual-derived midbrain neural precursor cells. Characterization of gene expression and metabolic modeling reveal pyruvate, several amino acid and lipid metabolism as the most dysregulated metabolic pathways in IPD neural precursors. Furthermore, we show that IPD neural precursors endure mitochondrial metabolism impairment and a reduced total NAD pool. Accordingly, we show that treatment with NAD precursors increases ATP yield hence demonstrating a potential to rescue early IPD-associated metabolic changes.

In mammals, new neurons are generated throughout the entire lifespan in two restricted areas of the brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ)-olfactory bulb (OB) system. In both regions newborn neurons display unique properties that clearly distinguish them from mature neurons. Enhanced excitability and increased synaptic plasticity enables them to add specific properties to information processing by modulating the existing local circuitry of already established mature neurons. Hippocampal neurogenesis has been suggested to play a role in spatial-navigation learning, spatial memory, and spatial pattern separation. Cumulative evidences implicate that adult-born OB neurons contribute to learning processes and odor memory. We recently demonstrated that the cell fate determinant TRIM32 is upregulated in differentiating neuroblasts of the SVZ-OB system in the adult mouse brain. The absence of TRIM32 leads to increased progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated OB neurons. Here, we present novel data from behavioral studies showing that such an enhancement of OB neurogenesis not necessarily leads to increased olfactory performance but in contrast even results in impaired olfactory capabilities. In addition, we show at the cellular level that TRIM32 protein levels increase during differentiation of neural stem cells (NSCs). At the molecular level, several metabolic intermediates that are connected to glycolysis, glycine, or cysteine metabolism are deregulated in TRIM32 knockout mice brain tissue. These metabolomics pathways are directly or indirectly linked to anxiety or depression like behavior. In summary, our study provides comprehensive data on how the impairment of neurogenesis caused by the loss of the cell fate determinant TRIM32 causes a decrease of olfactory performance as well as a deregulation of metabolomic pathways that are linked to mood disorders.

Aging is a complex trait of broad scientific interest, especially because of its intrinsic link with common human diseases. Pioneering work on aging-related mechanisms has been made in Saccharomyces cerevisiae, mainly through the use of deletion collections isogenic to the S288c reference strain. In this study, using a recently published high-throughput approach, we quantified chronological life span (CLS) within a collection of 58 natural strains across seven different conditions. We observed a broad aging variability suggesting the implication of diverse genetic and environmental factors in chronological aging control. Two major Quantitative Trait Loci (QTLs) were identified within a biparental population obtained by crossing two natural isolates with contrasting aging behavior. Detection of these QTLs was dependent upon the nature and concentration of the carbon sources available for growth. In the first QTL, the RIM15 gene was identified as major regulator of aging under low glucose condition, lending further support to the importance of nutrient-sensing pathways in longevity control under calorie restriction. In the second QTL, we could show that the SER1 gene, encoding a conserved aminotransferase of the serine synthesis pathway not previously linked to aging, is causally associated with CLS regulation, especially under high glucose condition. These findings hint toward a new mechanism of life span control involving a trade-off between serine synthesis and aging, most likely through modulation of acetate and trehalose metabolism. More generally it shows that genetic linkage studies across natural strains represent a promising strategy to further unravel the molecular basis of aging.

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

Neurodegeneration is a multistep process characterized by a multitude of molecular entities and their interactions. Systems analyses, or omics approaches, have become an important tool in characterizing this process. Although RNA and protein profiling made their entry into this field a couple of decades ago, metabolite profiling is a more recent addition. The metabolome represents a large part or all metabolites in a tissue, and gives a snapshot of its physiology. By using gas chromatography coupled to mass spectrometry, we analyzed the metabolic profile of brain regions of the mouse, and found that each region is characterized by its own metabolic signature. We then analyzed the metabolic profile of the mouse brain after excitotoxic injury, a mechanism of neurodegeneration implicated in numerous neurological diseases. More important, we validated our findings by measuring, histologically and molecularly, actual neurodegeneration and glial response. We found that a specific global metabolic signature, best revealed by machine learning algorithms, rather than individual metabolites, was the most robust correlate of neuronal injury and the accompanying gliosis, and this signature could serve as a global biomarker for neurodegeneration. We also observed that brain lesioning induced several metabolites with neuroprotective properties. Our results deepen the understanding of metabolic changes accompanying neurodegeneration in disease models, and could help rapidly evaluate these changes in preclinical drug studies.

Itaconic acid is produced by mammalian leukocytes upon pro-inflammatory activation. It appears to inhibit bacterial growth and to rewire the metabolism of the host cell by inhibiting succinate dehydrogenase. Yet, it is unknown whether itaconic acid acts only intracellularly, locally in a paracrine fashion, or whether it is even secreted from the inflammatory cells at meaningful levels in peripheral blood of patients with severe inflammation or sepsis. The aim of this study was to determine the release rate of itaconic acid from pro-inflammatory activated macrophages in vitro and to test for the abundance of itaconic acid in bodyfluids of patients suffering from acute inflammation. We demonstrate that excretion of itaconic acid happens at a low rate and that it cannot be detected in significant amounts in plasma or urine of septic patients or in liquid from bronchial lavage of patients with pulmonary inflammation. We conclude that itaconic acid may serve as a pro-inflammatory marker in immune cells but that it does not qualify as a biomarker in the tested body fluids.

Stem cell fate decisions are controlled by a molecular network in which transcription factors and miRNAs are of key importance. To systemically investigate their impact on neural stem cell (NSC) maintenance and neuronal commitment, we performed a high-throughput mRNA and miRNA profiling and isolated functional interaction networks of involved mechanisms. Thereby, we identified an E2F1-miRNA feedback loop as important regulator of NSC fate decisions. Although E2F1 supports NSC proliferation and represses transcription of miRNAs from the miR-17∼92 and miR-106a∼363 clusters, these miRNAs are transiently up-regulated at early stages of neuronal differentiation. In these early committed cells, increased miRNAs expression levels directly repress E2F1 mRNA levels and inhibit cellular proliferation. In mice, we demonstrated that these miRNAs are expressed in the neurogenic areas and that E2F1 inhibition represses NSC proliferation. The here presented data suggest a novel interaction mechanism between E2F1 and miR-17∼92 / miR-106a∼363 miRNAs in controlling NSC proliferation and neuronal differentiation.

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein-protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.

Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.