Pharmacological inhibition of MERTK induces in vivo retinal degeneration: a multimodal imaging ocular safety assessment

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Combined Deficiency of the Melanocortin 5 Receptor and Adenosine 2A Receptor Unexpectedly Provides Resistance to Autoimmune Disease in a CD8+ T Cell-Dependent Manner

Regulatory immunity that provides resistance to relapse emerges during resolution of experimental autoimmune uveitis (EAU). This post-EAU regulatory immunity requires a melanocortin 5 receptor (MC5r)-dependent suppressor antigen presenting cell (APC), as shown using a MC5r single knock-out mouse. The MC5r-dependent APC activates an adenosine 2A receptor (A2Ar)-dependent regulatory Treg cell, as shown using an A2Ar single knock-out mouse. Unexpectedly, when MC5r-/- post-EAU APC were used to activate A2Ar-/- post-EAU T cells the combination of cells significantly suppressed EAU, when transferred to EAU mice. In contrast, transfer of the reciprocal activation scheme did not suppress EAU. In order to explain this finding, MC5r-/-A2Ar-/- double knock-out (DKO) mice were bred. Naïve DKO mice had no differences in the APC populations, or inflammatory T cell subsets, but did have significantly more Treg cells. When we examined the number of CD4 and CD8 T cell subsets, we found significantly fewer CD8 T cells in the DKO mice compared to WT and both single knock-out mice. DKO mice also had significantly reduced EAU severity and accelerated resolution. In order to determine if the CD8 T cell deficiency contributed to the resistance to EAU in the DKO mice, we transferred naïve CD8 T cells from WT mice, that were immunized for EAU. Susceptibility to EAU was restored in DKO mice that received a CD8 T cell transfer. While the mechanism that contributed to the CD8 T cell deficiency in the DKO mice remains to be determined, these observations indicate an importance of CD8 T cells in the initiation of EAU. The involvement of CD4 and CD8 T cells suggests that both class I and class II antigen presentation can trigger an autoimmune response, suggesting a much wider range of antigens may trigger autoimmune disease.

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Deletion of the AMPylase mFICD alters cytokine secretion and affects cognitive plasticity in vivo

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This post-translational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mFICD AMPylase knock-out mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well-tolerated in unstressed mice. We show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM and IL-1β without affecting the unfolded protein response. Finally, we demonstrate that older mFICD-/- mice show improved cognitive plasticity. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.

Cyclin M2 (CNNM2) knockout mice show mild hypomagnesaemia and developmental defects

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/− mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2−/− pups were born alive. The Cnnm2−/− pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/− mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/− mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/− mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

Epigenomic profiling of stem cells within the pilosebaceous unit identifies PRDM16 as a regulator of sebaceous gland homeostasis

The epidermis consists of different compartments such as the hair follicle (HF), sebaceous gland (SG) and interfollicular epidermis (IFE), each containing distinct stem cell (SC) populations. However, with the exception of the SCs residing within the HF bulge, other epidermal SC populations remain less well understood. Here we used an epigenomic strategy that combines H3K27me3 ChIP-seq and RNA-seq profiling to identify major regulators of pilosebaceous unit (PSU) SC located outside the bulge. When applied to the bulk of PSU SC isolated from mouse skin our approach identified both previously known and potentially novel non-bulge PSU SC regulators. Among the latter, we found that PRDM16 was predominantly enriched within the Junctional Zone (JZ), which harbors SC that contribute to renewal of the upper HF and the SG. To investigate PRDM16 function in the PSU SC, we generated an epidermal-specific Prdm16 Knock-out mouse model (K14-Cre-Prdm16fl/fl). Notably, SG homeostasis was disturbed upon loss of PRDM16 resulting in enlarged SGs, and excessive sebum production, resembling some of the features associated with human acne and sebaceous hyperplasia. Importantly, PRDM16 is essential to shut down proliferation in differentiating sebocytes. Overall, our study provides a list of putative novel regulators of PSU SC outside the bulge and identifies PRDM16 as a major regulator of SG homeostasis.