A mutation in transmembrane protein 135 impairs lipid metabolism in mouse eyecups

Aging is a significant factor in the development of age-related diseases but how aging disrupts cellular homeostasis to cause age-related retinal disease is unknown. Here, we further our studies on transmembrane protein 135 (Tmem135), a gene involved in retinal aging, by examining the transcriptomic profiles of wild-type, heterozygous and homozygous Tmem135 mutant posterior eyecup samples through RNA sequencing (RNA-Seq). We found significant gene expression changes in both heterozygous and homozygous Tmem135 mutant mouse eyecups that correlate with visual function deficits. Further analysis revealed that expression of many genes involved in lipid metabolism are changed due to the Tmem135 mutation. Consistent with these changes, we found increased lipid accumulation in mutant Tmem135 eyecup samples. Since mutant Tmem135 mice have similar ocular pathologies as human age-related macular degeneration (AMD) eyes, we compared our homozygous Tmem135 mutant eyecup RNA-Seq dataset with transcriptomic datasets of human AMD donor eyes. We found similar changes in genes involved in lipid metabolism between the homozygous Tmem135 mutant eyecups and AMD donor eyes. Our study suggests that the Tmem135 mutation affects lipid metabolism as similarly observed in human AMD eyes, thus Tmem135 mutant mice can serve as a good model for the role of dysregulated lipid metabolism in AMD.

The Two β-Arrestins Regulate Distinct Metabolic Processes: Studies with Novel Mutant Mouse Models

The two β-arrestins (β-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G protein-coupled receptors. However, β-arrestins can also act as signaling molecules in their own right. Although the two proteins share a high degree of sequence and structural homology, early studies with cultured cells indicated that β-arrestin-1 and -2 are not functionally redundant. Recently, the in vivo metabolic roles of the two β-arrestins have been studied using mutant mice selectively lacking either β-arrestin-1 or -2 in cell types that are of particular relevance for regulating glucose and energy homeostasis. These studies demonstrated that the β-arrestin-1 and -2 mutant mice displayed distinct metabolic phenotypes in vivo, providing further evidence for the functional heterogeneity of these two highly versatile signaling proteins.

Rpl24Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The Rpl24Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that Rpl24Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, Apc and Kras. In contrast to previous reports, Rpl24Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the Rpl24Bst mutation has a tumour suppressive effect in mouse models.

Npr2 mutant mice show vasodilation and undeveloped adipocytes in mesentery

Objective The biological importance for the signaling of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) has been recognized. However, the details remain unclear and are debatable. The Npr2 is a gene of NPR-B, and we previously reported a unique phenotype of a spontaneous mutant mouse lacking Npr2 (Npr2slw/slw), such as severe ileus-like disorder with bloodless blood vessels. In this study, we analyzed the bloodless mesenteric vascular morphology of Npr2slw/slw by histological observation to clarify the effects of the CNP/NPR-B signal deficiency. Results Blood vessels in the mesentery were clearly dilated in the preweaning Npr2slw/slw mice. Additionally, in the Npr2slw/slw mice, the lacteals were partially dilation or randomly direction mucosal epithelial cells in villi, and mesenteric adipocytes were undeveloped. These findings provide important information for understanding the role of CNP/NPR-B signals on intestine with mesentery.

Progress towards completing the mutant mouse null resource

The generation of a comprehensive catalog of null alleles covering all protein-coding genes is the goal of the International Mouse Phenotyping Consortium. Over the past 20 years, significant progress has been made towards achieving this goal through the combined efforts of many large-scale programs that built an embryonic stem cell resource to generate knockout mice and more recently employed CRISPR/Cas9-based mutagenesis to delete critical regions predicted to result in frameshift mutations, thus, ablating gene function. The IMPC initiative builds on prior and ongoing work by individual research groups creating gene knockouts in the mouse. Here, we analyze the collective efforts focusing on the combined null allele resource resulting from strains developed by the research community and large-scale production programs. Based upon this pooled analysis, we examine the remaining fraction of protein-coding genes focusing on clearly defined mouse–human orthologs as the highest priority for completing the mutant mouse null resource. In summary, we find that there are less than 3400 mouse–human orthologs remaining in the genome without a targeted null allele that can be further prioritized to achieve our overall goal of the complete functional annotation of the protein-coding portion of a mammalian genome.

Deciphering the Biological Significance of ADAR1–Z-RNA Interactions

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi–Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation.

Increased Sociability in Mice Lacking Intergenic Dlx Enhancers

The Dlx homeodomain transcription factors play important roles in the differentiation and migration of GABAergic interneuron precursors. The mouse and human genomes each have six Dlx genes organized into three convergently transcribed bigene clusters (Dlx1/2, Dlx3/4, and Dlx5/6) with cis-regulatory elements (CREs) located in the intergenic region of each cluster. Amongst these, the I56i and I12b enhancers from the Dlx1/2 and Dlx5/6 locus, respectively, are active in the developing forebrain. I56i is also a binding site for GTF2I, a transcription factor whose function is associated with increased sociability and Williams–Beuren syndrome. In determining the regulatory roles of these CREs on forebrain development, we have generated mutant mouse-lines where Dlx forebrain intergenic enhancers have been deleted (I56i(–/–), I12b(–/–)). Loss of Dlx intergenic enhancers impairs expression of Dlx genes as well as some of their downstream targets or associated genes including Gad2 and Evf2. The loss of the I56i enhancer resulted in a transient decrease in GABA+ cells in the developing forebrain. The intergenic enhancer mutants also demonstrate increased sociability and learning deficits in a fear conditioning test. Characterizing mice with mutated Dlx intergenic enhancers will help us to further enhance our understanding of the role of these Dlx genes in forebrain development.

Analysis of amyloid-like secondary structure in the Cryab-R120G knock-in mouse model of hereditary cataracts by two-dimensional infrared spectroscopy

αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer’s, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.