Restoring cellular copper homeostasis in Alzheimer disease: a novel peptide shuttle is internalized by an ATP-dependent endocytosis pathway involving Rab5- and Rab14-endosomes.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aß peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.

Characterization of the Zebrafish Elastin a (elnasa12235) Mutant: A New Model of Elastinopathy Leading to Heart Valve Defects.

Elastic fibers are extracellular macromolecules that provide resilience and elastic recoil to elastic tissues and organs in vertebrates. They are composed of an elastin core surrounded by a mantle of fibrillin-rich microfibrils and are essentially produced during a relatively short period around birth in mammals. Thus, elastic fibers have to resist many physical, chemical, and enzymatic constraints occurring throughout their lives, and their high stability can be attributed to the elastin protein. Various pathologies, called elastinopathies, are linked to an elastin deficiency, such as non-syndromic supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS), and autosomal dominant cutis laxa (ADCL). To understand these diseases, as well as the aging process related to elastic fiber degradation, and to test potential therapeutic molecules in order to compensate for elastin impairments, different animal models have been proposed. Considering the many advantages of using zebrafish, we here characterize a zebrafish mutant for the elastin a paralog (elnasa12235) with a specific focus on the cardiovascular system and highlight premature heart valve defects at the adult stage.

Physiological Impact of a Synthetic Elastic Protein in Arterial Diseases Related to Alterations of Elastic Fibers: Effect on the Aorta of Elastin-Haploinsufficient Male and Female Mice.

Elastic fibers, made of elastin (90%) and fibrillin-rich microfibrils (10%), are the key extracellular components, which endow the arteries with elasticity. The alteration of elastic fibers leads to cardiovascular dysfunctions, as observed in elastin haploinsufficiency in mice (Eln+/-) or humans (supravalvular aortic stenosis or Williams-Beuren syndrome). In Eln+/+ and Eln+/- mice, we evaluated (arteriography, histology, qPCR, Western blots and cell cultures) the beneficial impact of treatment with a synthetic elastic protein (SEP), mimicking several domains of tropoelastin, the precursor of elastin, including hydrophobic elasticity-related domains and binding sites for elastin receptors. In the aorta or cultured aortic smooth muscle cells from these animals, SEP treatment induced a synthesis of elastin and fibrillin-1, a thickening of the aortic elastic lamellae, a decrease in wall stiffness and/or a strong trend toward a reduction in the elastic lamella disruptions in Eln+/- mice. SEP also modified collagen conformation and transcript expressions, enhanced the aorta constrictive response to phenylephrine in several animal groups, and, in female Eln+/- mice, it restored the normal vasodilatory response to acetylcholine. SEP should now be considered as a biomimetic molecule with an interesting potential for future treatments of elastin-deficient patients with altered arterial structure/function.

Quantification of cell contractile behavior based on non-destructive macroscopic measurement of tension forces on bioprinted hydrogel.

Contraction assay based on surface measurement have been widely used to evaluate cell contractility in 3D models. This method is straightforward and requires no specific equipment, but it does not provide quantitative data about contraction forces generated by cells. We expanded this method with a new biomechanical model, based on the work-energy theorem, to provide non-destructive longitudinal monitoring of contraction forces generated by cells in 3D. We applied this method on hydrogels seeded with either fibroblasts or osteoblasts. Hydrogel mechanical characteristics were modulated to enhance (condition HCAHigh: hydrogel contraction assay high contraction) or limit (condition HCALow: hydrogel contraction assay low contraction) cell contractile behaviors. Macroscopic measures were further correlated with cell contractile behavior and descriptive analysis of their physiology in response to different mechanical environments. Fibroblasts and osteoblasts contracted their matrix up to 47% and 77% respectively. Contraction stress peaked at day 5 with 1.1 10-14 Pa for fibroblasts and 3.5 10-14 Pa for osteoblasts, which correlated with cell attachment and spreading. Negligible contraction was seen in HCALow. Both fibroblasts and osteoblasts expressed α-SMA contractile fibers in HCAHigh and HCALow. Failure to contract HCALow was attributed to increased cross-linking and resistance to proteolytic degradation of the hydrogel.

MiR-30a-5p Alters Epidermal Terminal Differentiation during Aging by Regulating BNIP3L/NIX-Dependent Mitophagy.

Chronological aging is characterized by an alteration in the genes' regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction in BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decreasing with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.

Zebrafish as a Model to Study Vascular Elastic Fibers and Associated Pathologies.

Many extensible tissues such as skin, lungs, and blood vessels require elasticity to function properly. The recoil of elastic energy stored during a stretching phase is provided by elastic fibers, which are mostly composed of elastin and fibrillin-rich microfibrils. In arteries, the lack of elastic fibers leads to a weakening of the vessel wall with an increased risk to develop cardiovascular defects such as stenosis, aneurysms, and dissections. The development of new therapeutic molecules involves preliminary tests in animal models that recapitulate the disease and whose response to drugs should be as close as possible to that of humans. Due to its superior in vivo imaging possibilities and the broad tool kit for forward and reverse genetics, the zebrafish has become an important model organism to study human pathologies. Moreover, it is particularly adapted to large scale studies, making it an attractive model in particular for the first steps of investigations. In this review, we discuss the relevance of the zebrafish model for the study of elastic fiber-related vascular pathologies. We evidence zebrafish as a compelling alternative to conventional mouse models.

Development of thymic tumor in [LSL:KrasG12D; Pdx1-CRE] mice, an adverse effect associated with accelerated pancreatic carcinogenesis.

Pancreatic Ductal AdenoCarcinoma (PDAC) represents about 90% of pancreatic cancers. It is one of the most aggressive cancer, with a 5-year survival rate below 10% due to late diagnosis and poor therapeutic efficiency. This bad prognosis thus encourages intense research in order to better understand PDAC pathogenesis and molecular basis leading to the development of innovative therapeutic strategies. This research frequently involves the KC (LSL:KrasG12D;Pdx1-CRE) genetically engineered mouse model, which leads to pancreatic cancer predisposition. However, as frequently encountered in animal models, the KC mouse model also exhibits biases. Herein, we report a new adverse effect of KrasG12D mutation in KC mouse model. In our hands, 10% of KC mice developed clinical signs reaching pre-defined end-points between 100- and 150-days post-parturition, and associated with large thymic mass development. Histological and genetic analyses of this massive thymus enabled us (1) to characterize it as a highly proliferative thymic lymphoma and (2) to detect the unexpected recombination of the Lox-STOP-Lox cassette upstream KrasG12D allele and subsequent KRASG12D protein expression in all cells composing thymic masses. Finally, we highlighted that development of such thymic tumor was associated with accelerated pancreatic carcinogenesis, immune compartment disorganization, and in some cases, lung malignancies.

Advanced Characterization of Imiquimod-Induced Psoriasis-Like Mouse Model.

Many autoimmune disorders such as psoriasis lead to the alteration of skin components which generally manifests as unwanted topical symptoms. One of the most widely approved psoriasis-like animal models is the imiquimod (IMQ)-induced mouse model. This representation mimics various aspects of the complex cutaneous pathology and could be appropriate for testing topical treatment options. We perform a thorough characterization of this model by assessing some parameters that are not fully described in the literature, namely a precise description of skin disruption. It was evaluated by transepidermal water loss measurements and analyses of epidermis swelling as a consequence of keratinocyte hyperproliferation. The extent of neo-angiogenesis and hypervascularity in dermis were highlighted by immunostaining. Moreover, we investigated systemic inflammation through cytokines levels, spleen swelling and germinal centers appearance in draining lymph nodes. The severity of all parameters was correlated to IMQ concentration in skin samples. This study outlines new parameters of interest useful to assess this model. We highlight the skin barrier disruption and report a systemic inflammatory reaction occurring at distance both in spleen and lymph nodes. These newly identified biological endpoints could be exploited to investigate the efficacy of therapeutic candidates for psoriasis and more extensively for several other skin inflammatory diseases.

BMP-1 disrupts cell adhesion and enhances TGF-ß activation through cleavage of the matricellular protein thrombospondin-1.

Bone morphogenetic protein 1 (BMP-1) is an important metalloproteinase that synchronizes growth factor activation with extracellular matrix assembly during morphogenesis and tissue repair. The mechanisms by which BMP-1 exerts these effects are highly context dependent. Because BMP-1 overexpression induces marked phenotypic changes in two human cell lines (HT1080 and 293-EBNA cells), we investigated how BMP-1 simultaneously affects cell-matrix interactions and growth factor activity in these cells. Increasing BMP-1 led to a loss of cell adhesion that depended on the matricellular glycoprotein thrombospondin-1 (TSP-1). BMP-1 cleaved TSP-1 between the VWFC/procollagen-like domain and the type 1 repeats that mediate several key TSP-1 functions. This cleavage induced the release of TSP-1 C-terminal domains from the extracellular matrix and abolished its previously described multisite cooperative interactions with heparan sulfate proteoglycans and CD36 on HT1080 cells. In addition, BMP-1-dependent proteolysis potentiated the TSP-1-mediated activation of latent transforming growth factor-ß (TGF-ß), leading to increased signaling through the canonical SMAD pathway. In primary human corneal stromal cells (keratocytes), endogenous BMP-1 cleaved TSP-1, and the addition of exogenous BMP-1 enhanced cleavage, but this had no substantial effect on cell adhesion. Instead, processed TSP-1 promoted the differentiation of keratocytes into myofibroblasts and stimulated production of the myofibroblast marker α-SMA, consistent with the presence of processed TSP-1 in human corneal scars. Our results indicate that BMP-1 can both trigger the disruption of cell adhesion and stimulate TGF-ß signaling in TSP-1-rich microenvironments, which has important potential consequences for wound healing and tumor progression.

Complex regulation of two target genes encoding SPX-MFS proteins by rice miR827 in response to phosphate starvation.

Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.