Mechanism of action of phthalazinone derivatives against rabies virus.

Rabies, a viral zoonosis, is responsible for almost 59,000 deaths each year, despite the existence of an effective post-exposure prophylaxis. Indeed, rabies causes acute encephalomyelitis, with a case-fatality rate of 100 % after the onset of neurological clinical signs. Therefore, the development of therapies to inhibit the rabies virus (RABV) is crucial. Here, we identified, from a 30,000 compound library screening, phthalazinone derivative compounds as potent inhibitors of RABV infection and more broadly of Lyssavirus and even Mononegavirales infections. Combining in vitro experiments, structural modelling, in silico docking and in vivo assays, we demonstrated that phthalazinone derivatives display a strong inhibition of lyssaviruses infection by acting directly on the replication complex of the virus, and with noticeable effects in delaying the onset of the clinical signs in our mouse model.

Senescent melanocytes driven by glycolytic changes are characterized by melanosome transport dysfunction.

Rationale: Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A better understanding of the molecular characteristics of senescent melanocytes may be the key to controlling skin aging. Methods: We have developed an in vitro model of senescence in melanocytes using UV irradiation and investigated the functional characteristics and molecular mechanisms underlying senescence in UV-irradiated melanocytes. Results: We have highlighted that in vitro senescent melanocytes are characterized by melanosome transport dysfunction resulting in melanin accumulation. The defective melanosome transport was confirmed with the ultrastructural characterization of both in vitro UV-induced senescent melanocytes and in vivo melanocytes of hypopigmented aging skin. A single-cell transcriptomic analysis revealed that the glycolytic metabolism pathway appeared to be significantly upregulated in most senescent phenotypes. Furthermore, the inhibition of glycolysis by pharmacological compounds mitigates the pro-aging effects of melanocytes senescence, suggesting that alterations in cellular glucose metabolism act as a driving force for senescence in melanocytes. Conclusion: These results demonstrate that senescent melanocytes are characterized by glycolytic metabolism changes and a defective melanosome transport process, which may be related to impaired mitochondrial function, highlighting the importance of metabolic reprogramming in regulating melanocyte senescence.

PGC-1s shape epidermal physiology by modulating keratinocyte proliferation and terminal differentiation.

Skin plays central roles in systemic physiology, and it undergoes significant functional changes during aging. Members of the peroxisome proliferator-activated receptor-gamma coactivator (PGC-1) family (PGC-1s) are key regulators of the biology of numerous tissues, yet we know very little about their impact on skin functions. Global gene expression profiling and gene silencing in keratinocytes uncovered that PGC-1s control the expression of metabolic genes as well as that of terminal differentiation programs. Glutamine emerged as a key substrate promoting mitochondrial respiration, keratinocyte proliferation, and the expression of PGC-1s and terminal differentiation programs. Importantly, gene silencing of PGC-1s reduced the thickness of a reconstructed living human epidermal equivalent. Exposure of keratinocytes to a salicylic acid derivative potentiated the expression of PGC-1s and terminal differentiation genes and increased mitochondrial respiration. Overall, our results show that the PGC-1s are essential effectors of epidermal physiology, revealing an axis that could be targeted in skin conditions and aging.

Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models.

Organotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.

Pyrimethamine inhibits rabies virus replication in vitro.

Rabies virus transmits from animals to humans and causes encephalitis. Every year more than 15 million people receive a post exposure prophylaxis (PEP) treatment that is highly effective in the prevention of rabies disease. However, when clinical symptoms appear, for example in people who did not receive PEP, rabies is almost invariably fatal. Due to the limited access to PEP in some target populations, mostly in Asia and in Africa, rabies causes at least 59,000 deaths a year. PEP is not effective after the onset of symptoms and attempts to develop a treatment for clinical rabies have been unsuccessful. After screening a library of 385 FDA-approved drugs, we found that pyrimethamine inhibits rabies infection in vitro through the inhibition of adenosine synthesis. In addition, this compound shows a synergistic interaction with ribavirin. Unfortunately, in rabies infected-mice, pyrimethamine showed no efficacy. One possible explanation may be that the antiviral effect is negated by the observed interference of pyrimethamine with the innate immune response.

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis.

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBß-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNß, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

The matrix protein of rabies virus binds to RelAp43 to modulate NF-κB-dependent gene expression related to innate immunity.

The matrix (M) protein of wild isolates of rabies virus such as Tha (M-Tha) was previously shown to be able to interact with RelAp43, a protein of the NF-κB family, and to efficiently suppress NF-κB-dependent reporter gene expression, in contrast with the vaccine strain SAD. Here, we analyze the mechanisms involved in RelAp43-M protein interaction. We demonstrate that the central part of M-Tha, and the specific C-terminal region of RelAp43 are required for this interaction. Four differences in the corresponding amino acid sequences of the M-Tha and M-SAD are shown to be crucial for RelAp43 interaction and subsequent modulation of innate immune response. Furthermore, the capacity of M-Tha to interact with RelAp43 was shown to be crucial for the control of the expression of four genes (IFN, TNF, IL8 and CXCL2) during viral infection. These findings reveal that RelAp43 is a potent regulator of transcription of genes involved in innate immune response during rabies virus infection and that the M protein of wild isolates of rabies virus is a viral immune-modulatory factor playing an important role in this RelAp43-mediated host innate immunity response in contrast to M protein of vaccine strains, which have lost this property.

The human papillomavirus E6 oncogene represses a cell adhesion pathway and disrupts focal adhesion through degradation of TAp63ß upon transformation.

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63ß isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63ß is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and ß. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63ß although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63ß in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63ß is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63ß therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63ß in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.

A pivotal role for CXCL12 signaling in HPV-mediated transformation of keratinocytes: clues to understanding HPV-pathogenesis in WHIM syndrome.

The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.

A new E6/P63 pathway, together with a strong E7/E2F mitotic pathway, modulates the transcriptome in cervical cancer cells.

Cervical carcinoma is associated with certain types of human papillomaviruses expressing the E6 and E7 oncogenes, which are involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. A critical event on the path to malignant transformation is often manifested by the loss of expression of the viral E2 transcription factor due to the integration into the host genome of the viral DNA. Using microarrays, we have previously shown that reintroduction of a functional E2 in the HeLa cervical carcinoma cell line activates a cluster of p53 target genes while at the same time severely repressing a group of E2F target genes. In the present study, using new high-density microarrays containing more than 22,000 human cDNA sequences, we identified a novel p63 pathway among E2-activated genes and 38 new mitotic genes repressed by E2. We then sought to determine the pathways through which these genes were modulated and used an approach that relies on small interfering RNA to demonstrate that the p63 target genes were activated through silencing of the E6/E6AP pathway while the mitotic genes were mainly repressed through E7 silencing. Importantly, a subset of the mitotic genes was shown to be significantly induced in biopsies of stage IV cervical cancers, which points to a prominent E7 pathway in cervical carcinoma.