Biology subjects

Henriques, F.

Publications and source records attributed to Henriques, F..

8 recordsLinked to original sources

bioRxiv2023

Unravelling the regulatory function of a short 3'UTR sequence in zebrafish and in human cells

The mechanisms of gene expression regulation are essential for cell identity and function, and their disruption usually leads to human disease. The 3 untranslated region (UTR) of mRNA contains important regulatory elements of gene expression, including upstream sequence elements (USEs) that are cis-regulatory sequences localized upstream of polyA signals (PAS). One of the best functionally characterised USEs is located in the 3UTR of the Drosophilas polo gene, which disruption leads to critical phenotypes in adult flies. In this work we found that the USE of the Drosophilas polo gene (DplUSE) is also found in in the 3UTR of vertebrate genes, including zebrafish, mouse and human genes, showing higher levels of conservation than the whole 3UTR sequence. Using reporter assays, we show that DplUSE is able to increase gene expression in vitro in human cell lines and in vivo in zebrafish embryos. Importantly, in humans, the DplUSE containing genes are enriched for genes associated to serious diseases such as Congenital abnormalities and Malignant neoplasms, illustrating the potential of this sequence to modulate genes with relevant biological functions and related with human health. Concomitantly, when sequestering the molecular machinery that operates at the DplUSE using a dominant negative strategy, we show that this is enough to dysregulate DplUSE containing genes in human cells and disrupt proper embryo development in zebrafish. Aiming to understand the molecular mechanism operating at the DplUSE, we identified three RNA binding proteins (RBP) that specifically bind to the DplUSE in vertebrates. Importantly, one of such RBPs is PTBP1, the vertebrate orthologue of the fruit flys RBP Heph, that was demonstrated to be required for the DplUSE function in Drosophila. To test if PTBP1is essential for the DplUSE function, as observed in Drosophila, we depleted PTBP1 from human cells and observed a downregulation of the expression of DplUSE containing genes, demonstrating that the molecular mechanisms that operate at DplUSE are ultra-conserved. Finally, we explored if variants in DplUSE consensus could be associated to human disease. We found a reported single nucleotide polymorphism (SNP; rs3087967) that is associated with malignant tumor of colon and generates an ectopic consensus of DplUSE in the 3 UTR of the tumorigenic POU2AF2/C11orf53 gene. We further show that this ectopic DplUSE motif causes a gain-of-function in vivo in zebrafish gut cells, suggesting its involvement in colon cancer development. These results show that a short motif present in the 3UTR of genes from phylogenetically distant bilaterians, from fruit flies to humans, control genes expression through an ultra-conserved mechanism involving RBPs binding and its dysregulation might impact in human disease.

molecular biology↗

bioRxiv2023

Pathological beige remodeling induced by cancer cachexia depends on the disease severity and involves mainly the trans-differentiation of mature white adipocytes

In cancer associated cachexia (CAC), white adipose tissue undergoes morphofunctional and inflammatory changes that lead to tissue dysfunction and remodeling. In addition to metabolic changes in white adipose tissues (WAT), adipose tissue atrophy has been implicated in several clinical complications and poor prognoses associated with cachexia. Adipocyte atrophy may be associated with increased beige remodeling in human CAC as evidenced by the "beige remodeling" observed in preclinical models of CAC. Even though beige remodeling is associated with CAC-induced WAT dysfunction, there are still some open questions regarding their cellular origins. In this study, we investigated the development of beige remodeling in CAC from a broader perspective. In addition, we used a grading system to identify the scAT as being affected by mice weight loss early and intensely. Using different in vitro and ex-vivo techniques, we demonstrated that Lewis LLC1 cells can induce a switch from white to beige adipocytes, which is specific to this type of tumor cell. During the more advanced stages of CAC, beige adipocytes are mainly formed from the transdifferentiation of cells. According to our results, humanizing the CAC classification system is an efficient approach to defining the onset of the syndrome in a more homogeneous manner. Pathological beige remodeling occurred early in the disease course and exhibited phenotypic characteristics specific to LLC cells secretomes. Developing therapeutic strategies that recruit beige adipocytes in vivo may be better guided by an understanding of the cellular origins of beige adipocytes emitted by CAC.

cell biology↗

bioRxiv2022

De novo lipogenesis fuels adipocyte autophagosome membrane dynamics

Autophagy is a homeostatic degradative process for cell components that enables stress resilience and can determine cellular fate and function. However, lipid sources for the rapid membrane expansions of autophagosomes, the workhorses of autophagy, are poorly understood. Here, we identify de novo lipogenesis (DNL) as a critical source of fatty acids (FA) to fuel autophagosome dynamics in adipocytes. Adipocyte fatty acid synthase (Fasn) deficiency markedly impairs autophagy, evident by autophagosome accumulation, and severely compromises degradation of the autophagic substrate p62. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes even though lipid droplet size is restored. Imaging studies reveal that Fasn colocalizes with nascent autophagosomes, while loss of Fasn decreases certain membrane phosphoinositides known to be required for autophagosome assembly. Together, our studies highlight a newly appreciated function for adipocyte DNL in autophagosome membrane formation and provide evidence that localized FA synthesis contributes to autophagosome dynamics.

molecular biology↗

bioRxiv2020

CRISPR-enhanced human adipocyte 'browning' as cell therapy for metabolic disease

Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin1,2, disturbances in glucose and lipid fluxes3-5 and comorbidities including steatohepatitis6 and cardiovascular disease7,8. Despite extensive efforts at prevention and treatment9,10, diabetes afflicts over 400 million people worldwide11. Whole body metabolism is regulated by adipose tissue depots12-14, which include both lipid-storing white adipocytes and less abundant "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health15-18. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing19,20 to enhance "browning" of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP121,22 with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.

bioengineering↗

bioRxiv2020

Single Cell RNA Profiling Reveals Adipocyte to Macrophage Signaling Sufficient to Enhance Thermogenesis

The "browning" of inguinal white adipose tissue (iWAT) through increased abundance of thermogenic beige/brite adipocytes is induced by cold exposure and many other perturbations in association with beneficial systemic metabolic effects. Adipose browning is reported to require activation of sympathetic nerve fibers (SNF), aided by alternately activated macrophages within iWAT. Here we demonstrate the first example of a non-cell autonomous pathway for iWAT browning that is fully independent of SNF activity. Thus, the strong induction of thermogenic adipocytes prompted by deletion of adipocyte fatty acid synthase (iAdFASNKO mice) was unaffected by denervation or the deletion of SNF modulator Neuregulin-4. However, browning of iWAT in iAdFASNKO mice does require adipocyte cAMP/protein kinase A signaling, as it was blocked in adipocyte- selective Fasn/Gs double KO mice. Single-cell transcriptomic analysis of iAdFASNKO mouse adipose stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type. Mechanistically, depletion of such phagocytic immune cells in iAdFASNKO mice fully abrogated appearance of thermogenic adipocytes in iWAT. Altogether, these findings reveal an unexpected pathway of cAMP/PKA-dependent iWAT browning that is initiated by adipocyte signals and caused by macrophage-like cells independent of sympathetic neuron involvement.

physiology↗

bioRxiv2018

Toll-Like Receptor-4 Disruption Suppresses Adipose Tissue Remodeling and Increases Survival During Cancer Cachexia Syndrome

Cancer-induced cachexia, characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling and muscle wasting, is a malignant metabolic syndrome with undefined etiology. Here, we show that Toll-like receptor 4 (TLR4) mediates AT remodeling, in particular, AT browning and inflammatory response in mice bearing Lewis lung carcinoma (LLC). LLC tumor-bearing (TB) TLR4-/- mice were spared from AT remodeling due to a reduced macrophage infiltration and adipocyte atrophy. TLR4-/- mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 reproduced the main protective effect against AT remodeling found in TLR4-/- TB mice. Moreover, the treatment was effective in prolonging the survival and attenuating tumor mass growth when compared to non-treated-TB animals. Further, tumor-induced elevation of circulating pro-inflammatory cytokines was similarly abolished in both genetic ablation and pharmacological inhibition of TLR4. These data suggest that TLR4 is a critical mediator and a promising therapeutic target for cancer-induced AT remodeling.\n\nHIGHLIGHTSO_LIGenetic ablation and pharmacological inhibition of TLR4 attenuate adipose tissue remodeling during cancer-associated cachexia;\nC_LIO_LITLR4 suppression play an essential role in the browning phenotype induced by cachexia;\nC_LIO_LIAdministration of TLR4 drug inhibitor increase survival and reduces tumor mass growth in tumor bearing mice;\nC_LIO_LITLR4 pathway is a promising target for cancer-cachexia therapeutic intervention.\nC_LI

cancer biology↗

bioRxiv2018

CRISPR delivery particles for developing therapeutic strategies in metabolic disease

RNA-guided engineered nucleases derived from a prokaryotic adaptive immune system known as CRISPR-Cas represent a promising platform for gene deletion and editing. As a therapeutic approach, direct delivery of Cas9 protein and guide RNA could circumvent the safety problems associated with plasmid delivery and therefore represents an attractive tool for genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fat oxidation and secretion of bioactive factors through a \"browning\" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed novel CRISPR delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and single guide (sg)RNA, coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9 mediated gene deletion of ectopically expressed Green fluorescent protein (GFP) by CriPs was achieved in multiple cell types including a macrophage cell line, primary macrophages and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP expressing mice following intraperitoneal injection of CriPs containing sgRNA targeting Gfp. Furthermore, the disruption of the Nrip1 gene in white adipocytes by CriPs enhanced adipocyte \"browning\" with a marked increase of UCP1 expression. Deletion of Nrip1 by CriPs did not produce detectable off-target effects. Thus CriPs represent a novel CRISPR delivery system for Cas9 and sgRNA that is effective for ablating targeted gene products in cultured cells and in vivo, and provide a potential therapeutic strategy for metabolic disease.

bioengineering↗

bioRxiv2018

Neuronal Modulation of Brown Adipose Activity Through Perturbation of White Adipocyte Lipogenesis

White adipose tissue (WAT) secretes factors to communicate with other metabolic organs to maintain energy homeostasis. We previously reported that perturbation of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) causes expansion of sympathetic neurons within white adipose tissue (WAT) and the appearance of \"beige\" adipocytes. Here we report evidence that white adipocyte DNL activity is also coupled to neuronal regulation and thermogenesis in brown adipose tissue (BAT). Induced deletion of FASN in all adipocytes in mature mice (iAdFASNKO) enhanced sympathetic innervation and neuronal activity as well as UCP1 expression in both WAT and BAT. In contrast, selective ablation of FASN in brown adipocytes of mice (iUCP1FASNKO) failed to modulate sympathetic innervation and the thermogenic program in BAT. Surprisingly, DNL in brown adipocytes was also dispensable in maintaining euthermia when UCP1FASNKO mice were cold-exposed. These results indicate that DNL in white adipocytes influences long distance signaling to BAT, which can modify BAT sympathetic innervation and expression of genes involved in thermogenesis.

cell biology↗