N1-acetylspermidine is a determinant of hair follicle stem cell fate

Stem cell differentiation is accompanied by increased mRNA translation. The rate of protein biosynthesis is influenced by the polyamines putrescine, spermidine and spermine, which are essential for cell growth and stem cell maintenance. However, the role of polyamines as endogenous effectors of stem cell fate and whether they act through translational control remains obscure. Here, we investigate the function of polyamines in stem cell fate decisions using hair follicle stem cell (HFSC) organoids. Compared to progenitor cells, HFSCs showed lower translation rates, correlating with reduced polyamine levels. Surprisingly, overall polyamine depletion decreased translation but did not affect cell fate. In contrast, specific depletion of natural polyamines mediated by spermidine/spermine N1-acetyltransferase (SSAT; also known as SAT1) activation did not reduce translation but enhanced stemness. These results suggest a translation-independent role of polyamines in cell fate regulation. Indeed, we identified N1-acetylspermidine as a determinant of cell fate that acted through increasing self-renewal, and observed elevated N1-acetylspermidine levels upon depilation-mediated HFSC proliferation and differentiation in vivo. Overall, this study delineates the diverse routes of polyamine metabolism-mediated regulation of stem cell fate decisions. This article has an associated First Person interview with the first author of the paper.Peer reviewe

Metabolic control of hair follicle stem cell fate decisions

Aging is the main risk factor for the manifestation of numerous serious diseases, including cancer, cardiovascular diseases and neurodegeneration. Thus, the growing proportion of elderly people in the population poses a major challenge to the health care systems worldwide. Tissue homeostasis declines with age and its maintenance late in life has been intimately linked to prolonged healthspan. Stem cells act as drivers of tissue homeostasis through the interplay of self-renewal and differentiation. Therefore, it is of utmost importance to identify stem cell fate determinants to be able to delay stem cell exhaustion in the elderly. Of note, metabolic changes have been described to influence function and maintenance of stem cell populations. However, whether manipulation of metabolism affects cell fate decisions and stem cell maintenance remains poorly understood. Here, I focus on three metabolic pathways that have been described to become dysregulated with age, and to affect stemness: the hexosamine pathway (HP), aerobic sugar metabolism, and the polyamine metabolism. I assess their influence on cell fate decisions in an in vitro organoid culture system, which allows for the manipulation of hair follicle stem cells (HFSCs) and their direct progeny. First, I activate the HP by GlcNAc supplementation and through genetic manipulation of the rate-limiting enzyme GFAT1. While pathway flux and downstream metabolites increase, HFSC fate is not affected by these interventions. Second, I investigate the effect of sugar supplementation on cell fate decisions. Both, D-glucose and D-mannose, enhance stemness. While D-glucose supplementation boosts glycolysis, D-mannose addition specifically increases the acetylated polyamines N1-acetylspermidine (N1-AcSpd) and N1-acetylspermine without elevating glycolytic flux. Third, I elucidate how polyamine levels influence cell fate decisions. I confirm that low translation rates mark the stem cell state and that a forced decrease in translation is sufficient to elevate stemness. Surprisingly, I demonstrate that reducing translation by changes in polyamine availability does not correlate with increased stemness in the organoids. I identify N1-AcSpd as novel regulator of HFSC fate decisions, accelerating cell cycle progression. Finally, I demonstrate that HFSC activation by depilation results in an elevation of the acetylated polyamines, suggesting a functional role of N1-AcSpd in cell fate decisions in vivo. Overall, my results suggest that manipulation of metabolism is an effective means to control cell fate decisions, delay stem cell exhaustion, and improve tissue homeostasis in the elderly

The RATIOnal Role of Polyamines in Epidermal Differentiation

Polyamines have been implicated in skin tumorigenesis; however, their role in epidermal homeostasis remains obscure. In a new article in the Journal of Investigative Dermatology, Rahim et al. (2021) report that keratinocyte differentiation requires a shift in polyamine ratios that is mediated by AMD1. Results suggest that targeting polyamine availability might be useful in the treatment of hyperproliferative skin disorders

Hexosamine pathway activation improves memory but does not extend lifespan in mice

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5-diphospho-N-acetyl-d-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies. p id=Par Mutations in the hexosamine pathway key enzyme glutamine fructose-6-phosphate amidotransferase (GFAT-1) improve protein quality control and extend C. elegans lifespan. Here the authors present the crystal structures of full-length human GFAT-1 alone and with bound ligands and perform activity assays, which show that gain-of-function in the longevity-associated G451E variant is caused by a loss of feedback regulation

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5′-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies

Hexosamine Pathway Activation Improves Protein Homeostasis through the Integrated Stress Response

Activation of the hexosamine pathway (HP) through gain-of-function mutations in its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT-1) ameliorates proteotoxicity and increases lifespan in Caenorhabditis elegans. Here, we investigate the role of the HP in mammalian protein quality control. In mouse neuronal cells, elevation of HP activity led to phosphorylation of both PERK and eIF2 alpha as well as downstream ATF4 activation, identifying the HP as a modulator of the integrated stress response (ISR). Increasing uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) levels through GFAT1 gain-of-function mutations or supplementation with the precursor GlcNAc reduces aggregation of the polyglutamine (polyQ) protein Ataxin-3. Blocking PERK signaling or autophagy suppresses this effect. In C. elegans, overexpression of gfat-1 likewise activates the ISR. Consistently, co-overexpression of gfat-1 and proteotoxic polyQ peptides in muscles reveals a strong protective cell-autonomous role of the HP. Thus, the HP has a conserved role in improving protein quality control through modulation of the ISR

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP‑GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N‑acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP‑GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP