Functional invalidation of the autotaxin gene by a single amino acid mutation in mouse is lethal

AbstractAutotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX −/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX−/− mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015–5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822–25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies

Efficient generation of human immune system rats using human CD34+ cells

International audienceHuman immune system (HIS) mice generated using human CD34+ hematopoietic stem cells serve as a pivotal model for the in vivo evaluation of immunotherapies for humans. Yet, HIS mice possess certain limitations. Rats, due to their size and comprehensive immune system, hold promise for translational experiments. Here, we describe an efficacious method for long-term immune humanization, through intrahepatic injection of hCD34+ cells in newborn immunodeficient rats expressing human SIRPα. In contrast to HIS mice and similar to humans, HIS rats showed in blood a predominance of T cells, followed by B cells. Immune humanization was also high in central and secondary lymphoid organs. HIS rats treated with the anti-human CD3 antibody were depleted of human T cells, and human cytokines were detected in sera. We describe for the first time a method to efficiently generate HIS rats. HIS rats have the potential to be a useful model for translational immunology

Mistranslation-associated perturbations of proteostasis do not promote accumulation of amyloid beta and plaque deposition in aged mouse brain

A common perception in age-related neurodegenerative diseases posits that a decline in proteostasis is key to the accumulation of neuropathogenic proteins, such as amyloid beta (Aβ), and the development of sporadic Alzheimer's disease (AD). To experimentally challenge the role of protein homeostasis in the accumulation of Alzheimer's associated protein Aβ and levels of associated Tau phosphorylation, we disturbed proteostasis in single APP knock-in mouse models of AD building upon Rps9 D95N, a recently identified mammalian ram mutation which confers heightened levels of error-prone translation together with an increased propensity for random protein aggregation and which is associated with accelerated aging. We crossed the Rps9 D95N mutation into knock-in mice expressing humanized Aβ with different combinations of pathogenic mutations (wild-type, NL, NL-F, NL-G-F) causing a stepwise and quantifiable allele-dependent increase in the development of Aβ accumulation, levels of phosphorylated Tau, and neuropathology. Surprisingly, the misfolding-prone environment of the Rps9 D95N ram mutation did not affect Aβ accumulation and plaque formation, nor the level of phosphorylated Tau in any of the humanized APP knock-in lines. Our findings indicate that a misfolding-prone environment induced by error-prone translation with its inherent perturbations in protein homeostasis has little impact on the accumulation of pathogenic Aβ, plaque formation and associated phosphorylated Tau