Circadian Regulation of Adult Stem Cell Homeostasis and Aging

The circadian clock temporally organizes cellular physiology throughout the day, allowing daily environmental changes to be anticipated and potentially harmful physiologic processes to be temporally separated. By synchronizing all cells at the tissue level, the circadian clock ensures coherent temporal organismal physiology. Recent advances in our understanding of adult stem cell physiology suggest that aging and perturbations in circadian rhythmicity in stem cells are tightly intertwined. Here we discuss how circadian rhythms regulate and synchronize adult stem cell functions and how alterations in clock function during aging modulate the extrinsic and intrinsic mechanisms that determine adult stem cell homeostasis

In vivo analysis of the role of FADD in the regulation of intestinal epithelial homeostasis

The initiation of cell death can be an integral part of the immune response to infected or otherwise damaged cells. FADD is an adaptor protein connecting various immune response related signalling pathways to the induction of apoptotic cell death. FADD dependent signalling has mainly been studied in immune cells, while the in vivo role of FADD in epithelial tissues, which are at the border to various stress factors from outside the organism, is largely unknown. The intestinal epithelium is a single cell layer that forms a barrier, separating the luminal microbiota from the mucosal immune cells in the gastro-intestinal tract. The integrity of this barrier is essential for intestinal homeostasis, making the regulation of intestinal epithelial cell (IEC) death in response to infections or other stress-factors an important issue. In this thesis, the role of FADD in the intestinal epithelium was studied. We found that FADD is essential for intestinal homeostasis by preventing excessive RIP3 dependent IEC necrosis. Mice lacking FADD specifically in the intestinal epithelium (FADDIEC-KO mice) developed severe spontaneous colitis and enteritis. IEC necrosis in the colon of FADDIEC-KO mice was abrogated by deletion of RIP3 or by the expression of a CYLD mutant lacking the deubiquitinase activity. Like in the colon, necrosis of FADD deficient IECs in the small intestinal epithelium was also depending on RIP3. However, the deubiquitinating activity of CYLD was not required for small intestinal IEC necrosis to occur, suggesting different pathways to mediate IEC necrosis in the colon and small intestine of FADDIEC-KO mice. Development of colitis was partially depending on the expression of TNF and MyD88 mediated signalling induced by the microbiota. On the contrary, enteritis development in FADDIEC-KO mice was independent of TNF, the commensal bacteria and MyD88 dependent signalling. Thus, different mechanisms are responsible for the development of inflammation in the colon and small intestine of FADDIEC-KO mice. The results presented in this thesis demonstrate that FADD is not only a potential mediator of apoptotic cell death in the intestinal epithelium, but it is also essential for IEC survival under homeostatic conditions by preventing RIP3 dependent IEC necrosis

The tumor suppressor CYLD regulates the p53 DNA damage response

The tumour suppressor CYLD is a deubiquitinase previously shown to inhibit NF-κB, MAP kinase and Wnt signalling. However, the tumour suppressing mechanisms of CYLD remain poorly understood. Here we show that loss of CYLD catalytic activity causes impaired DNA damage-induced p53 stabilization and activation in epithelial cells and sensitizes mice to chemical carcinogen-induced intestinal and skin tumorigenesis. Mechanistically, CYLD interacts with and deubiquitinates p53 facilitating its stabilization in response to genotoxic stress. Ubiquitin chain-restriction analysis provides evidence that CYLD removes K48 ubiquitin chains from p53 indirectly by cleaving K63 linkages, suggesting that p53 is decorated with complex K48/K63 chains. Moreover, CYLD deficiency also diminishes CEP-1/p53-dependent DNA damage-induced germ cell apoptosis in the nematode Caenorhabditis elegans. Collectively, our results identify CYLD as a deubiquitinase facilitating DNA damage-induced p53 activation and suggest that regulation of p53 responses to genotoxic stress contributes to the tumour suppressor function of CYLD

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Collecting mouse livers for transcriptome analysis of daily rhythms

Molecular daily rhythms can be captured by precisely timed tissue harvests from groups of animals. This protocol will allow the investigator to identify transcriptional rhythms in the mouse liver while also providing a template for similar analyses in other whole metabolic organs. We describe steps for mouse entrainment, liver dissection, and rhythmicity analysis from total RNA sequencing data. The resulting rhythmic transcriptome will provide the user with a starting point for defining specific biological processes that undergo daily rhythms. For complete details on the use and execution of this protocol, please refer to Koronowski et al. (2019). A similar protocol for interfollicular epidermal cells is demonstrated in Welz et al. (2019).The IRB Barcelona is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). K.B.K. was supported by NIH-NIDDK (award F32DK121425). T.M. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510

TLR-independent anti-inflammatory function of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice

Objective: The gut microbiota modulates host susceptibility to intestinal inflammation, but the cell types and the signalling pathways orchestrating this bacterial regulation of intestinal homeostasis remain poorly understood. Here, we investigated the function of intestinal epithelial toll-like receptor (TLR) responses in the dextran sodium sulfate (DSS)-induced mouse model of colitis. Design: We applied an in vivo genetic approach allowing intestinal epithelial cell (IEC)-specific deletion of the critical TLR signalling adaptors, MyD88 and/or TIR-domain-containing adapter-inducing interferon-β (TRIF), as well as the downstream ubiquitin ligase TRAF6 in order to reveal the IEC-intrinsic function of these TLR signalling molecules during DSS colitis. Results: Mice lacking TRAF6 in IECs showed exacerbated DSS-induced inflammatory responses that ensued in the development of chronic colon inflammation. Antibiotic pretreatment abolished the increased DSS susceptibility of these mice, showing that epithelial TRAF6 signalling pathways prevent the gut microbiota from driving excessive colitis. However, in contrast to epithelial TRAF6 deletion, blocking epithelial TLR signalling by simultaneous deletion of MyD88 and TRIF specifically in IECs did not affect DSS-induced colitis severity. This in vivo functional comparison between TRAF6 and MyD88/TRIF deletion in IECs shows that the colitis-protecting effects of epithelial TRAF6 signalling are not triggered by TLRs. Conclusions: Intestinal epithelial TRAF6-dependent but MyD88/TRIF-independent and, thus, TLR-independent signalling pathways are critical for preventing propagation of DSS-induced colon inflammation by the gut microbiota. Moreover, our experiments using mice with dual MyD88/TRIF deletion in IECs unequivocally show that the gut microbiota trigger non-epithelial TLRs rather than epithelial TLRs to restrict DSS colitis severity

The Adaptor Protein FADD Protects Epidermal Keratinocytes from Necroptosis In Vivo and Prevents Skin Inflammation

Epidermal keratinocytes provide an essential structural and immunological barrier forming the first line of defense against potentially pathogenic microorganisms. Mechanisms regulating barrier integrity and innate immune responses in the epidermis are important for the maintenance of skin immune homeostasis and the pathogenesis of inflammatory skin diseases. Here, we show that epidermal keratinocyte-restricted deficiency of the adaptor protein FADD (FADD(E-KO)) induced severe inflammatory skin lesions in mice. The development of skin inflammation in FADD(E-KO) mice was triggered by RIP kinase 3 (RIP3)-mediated programmed necrosis (termed necroptosis) of FADD-deficient keratinocytes, which was partly dependent on the deubiquitinating enzyme CYLD and tumor necrosis factor (TNF)-TNF receptor 1 signaling. Collectively, our findings provide an in vivo experimental paradigm that regulation of necroptosis in keratinocytes is important for the maintenance of immune homeostasis and the prevention of chronic inflammation in the skin

Bmal1-knockout mice exhibit reduced cocaine-seeking behaviour and cognitive impairments

Brain and Muscle Arnt-like Protein 1 (BMAL1) is an essential component of the molecular clock underlying circadian rhythmicity. Its function has been recently associated with mood and reward processing alterations. We investigated the behavioural and neurobiological impact of Bmal1 gene deletion in mice, and how this could affect rewarding effects of cocaine. Additionally, key clock genes and components of the dopamine system were assessed in several brain areas. Our results evidence behavioural alterations in Bmal1-KO mice, including changes in locomotor activity with impaired habituation to environments, short-term memory and social recognition impairments. In addition, Bmal1-KO mice experienced reduced cocaine-induced sensitisation and rewarding effects of cocaine as well as reduced cocaine-seeking behaviour. Furthermore, Bmal1 deletion influenced the expression of other clock-related genes in the mPFC and striatum, as well as alterations in the expression of dopaminergic elements. Overall, the present article offers a novel and extensive characterisation of Bmal1-KO animals. We suggest that reduced cocaine's rewarding effects in these mutant mice might be related to Bmal1 role as an expression regulator of MAO and TH, two essential enzymes involved in dopamine metabolism.This study was supported by the Ministerio de Ciencia e Innovacion (grant number PID2019-104077RB-100/AEI/10.13039/501100011033), Ministerio de Sanidad (Retic-ISCIII, grant number RD16/017/010; RICORS, grant number RD21/0009/0001, and Plan Nacional sobre Drogas 2018/007). L.A.-Z. and I.G.-L. received a FPI grants (BES-2017-080066 and PRE2020-091923) from the Ministerio de Economia y Competividad. P.S.W. was supported by grant RYC2019-026661-I funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future". The Department of Medicine and Health Sciences (MELIS-UPF) is a “Unidad de Excelencia María de Maeztu” funded by the AEI (CEX2018-000792-M). O.V. is recipient of an ICREA Academia Award (Institució Catalana de Recerca i Estudis Avançats, Generalitat de Catalunya)