Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense mediated decay (NMD) is the cell's natural surveillance mechanism, that detects and destroys PTC containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using RT-qPCR, we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation (c.715 C > T; p.[R239*]). These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their iPSC-derived RPE. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wildtype levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated

Comparison of nonsense-mediated mRNA decay efficiency in various murine tissues

<p>Abstract</p> <p>Background</p> <p>The Nonsense-Mediated mRNA Decay (NMD) pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type <it>Men1 </it>transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed <it>Men1 </it>gene.</p> <p>Results</p> <p>Significant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type <it>Men1 </it>transcripts, identical to the value measured in murine embryonic fibroblasts). The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%). Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the <it>Men1 </it>gene.</p> <p>Conclusion</p> <p>Mouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.</p

Attenuation of IgG immune complex‐induced acute lung injury by silencing C5aR in lung epithelial cells

Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular‐airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell C5aR is up‐regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper‐airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs.—Sun, L., Guo, R.‐F., Gao, H., Sarma, J. V., Zetoune, F. S., Ward, P. A. Attenuation of IgG immune complex‐induced acute lung injury by silencing C5aR in lung epithelial cells. FASEB J. 23, 3808–3818 (2009). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154350/1/fsb2fj09133694.pd

Anti‐inflammatory effects of β2 adrenergic receptor agonists in experimental acute lung injury

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154493/1/fsb2026005038.pd

MyD88‐dependent production of IL‐17F is modulated by the anaphylatoxin C5a via the Akt signaling pathway

The interleukin‐17 (IL‐17) family of cytokines plays important roles in innate immune defenses against bacterial and fungal pathogens. While much is known about IL‐17A, much less information is available about the IL‐17F isoform. Here, we investigated gene expression and release of IL‐17F and its regulation by the complement system. IL‐17F was produced in mouse peritoneal elicited macrophages after TLR4 activation by LPS, peaking after 12 h. This effect was completely dependent on the presence of the adaptor protein MyD88. The copresence of the complement activation product, C5a (EC50=10 nM), amplified IL‐17F production via the receptor C5aR. In vitro signaling studies indicated that LPS or C5a, or the combination, caused phosphorylation of Akt occurring at threonine 308 but not at serine 473. Treatment of macrophages with pharmacologic inhibitors of PI3K‐Akt greatly reduced production of IL‐17F as well as mRNA for IL‐17F. In endotoxemia, C5a levels peaked at 6 h, while IL‐17F levels peaked between 6‐12 h. Full in vivo production of IL‐17F during endotoxemia required C5a. A similar result was found in the cecal ligation and puncture sepsis model. These data suggest that maximal production of IL‐17F requires complement activation and presence of C5a.—Bosmann, M., Patel, V. R., Russkamp, N. F., Pache, F., Zetoune, F. S., Sarma, J. V., Ward, P. A. MyD88‐dependent production of IL‐17F is modulated by the anaphylatoxin C5a via the Akt signaling pathway. FASEB J. 25, 4222–4232 (2011). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154335/1/fsb2fj11191205.pd

Extracellular histones are essential effectors of C5aR‐ and C5L2‐mediated tissue damage and inflammation in acute lung injury

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154331/1/fsb2027012034.pd

Functions of the complement components C3 and C5 during sepsis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154678/1/fsb2fj08110595.pd

Complementâ induced activation of the cardiac NLRP3 inflammasome in sepsis

Cardiac dysfunction develops during sepsis in humans and rodents. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), we investigated the role of the NLRP3 inflammasome in the heart. Mouse heart homogenates from shamâ procedure mice contained high mRNA levels of NLRP3 and ILâ 1β. Usingthe inflamm a some protocol, exposure of cardiomyocytes (CMs) to LPS followed by ATP or nigericin caused release of mature ILâ 1β. Immuno staining of left ventricular frozen sections before and 8 h after CLP revealed the presence of NLRP3 and ILâ 1β proteins inCMs. CLP caused substantial increases in mRNAs for ILâ 1β and NLRP3 in CMs which are reduced in the absence of either C5aR1 or C5aR2. After CLP, NLRP32/2 mice showed reduced plasma levels of ILâ 1βand ILâ 6. In vitro exposure of wildâ type CMs to recombinant C5a (rC5a) cause delevations in both cytosolic and nuclear/mitochondrial reactive oxygen species (ROS), which were C5aâ receptor dependent. Use of a selective NOX2 inhibitor prevented increased cytosolic and nuclear/mitochondrial ROS levels and release of ILâ 1β. Finally, NLRP32/2 mice had reduced defects in echo/Doppler parameters in heart afterCLP. These studies establish that the NLRP3 inflammasome contributes to the cardiomyopathy of polymicrobial sepsis.â Kalbitz, M., Fattahi, F., Grailer, J. J., Jajou, L., Malan, E. A., Zetoune, F. S., Huberâ Lang, M., Russell, M. W., Ward, P. A. Complementâ induced activation of the cardiac NLRP3 inflammasome in sepsis. FASEB J. 30, 3997â 4006 (2016). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154362/1/fsb2fasebj30120728r.pd

Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction

Polymicrobial sepsis in mice causes myocardial dysfunction after generation of the complement anaphylatoxin, complement component 5a (C5a). C5a interacts with its receptors on cardiomyocytes (CMs), resulting in redox imbalance and cardiac dysfunction that can be functionally measured and quantitated using Doppler echocardiography. In this report we have evaluated activation of MAPKs and Akt in CMs exposed to C5a in vitro and after cecal ligation and puncture (CLP) in vivo. In both cases, C5a in vitro caused activation (phosphorylation) of MAPKs and Akt in CMs, which required availability of both C5a receptors. Using immunofluorescence technology, activation of MAPKs and Akt occurred in left ventricular (LV) CMs, requiring both C5a receptors, C5aR1 and â 2. Use of a waterâ soluble p38 inhibitor curtailed activation in vivo of MAPKs and Akt in LV CMs as well as the appearance of cytokines and histones in plasma from CLP mice. When mouse macrophages were exposed in vitro to LPS, activation of MAPKs and Akt also occurred. The copresence of the p38 inhibitor blocked these activation responses. Finally, the presence of the p38 inhibitor in CLP mice reduced the development of cardiac dysfunction. These data suggest that polymicrobial sepsis causes cardiac dysfunction that appears to be linked to activation of MAPKs and Akt in heart.â Fattahi, F., Kalbitz, M., Malan, E. A., Abe, E., Jajou, L., Huberâ Lang, M. S., Bosmann, M., Russell, M. W., Zetoune, F. S., Ward, P. A. Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction. FASEB J. 31, 4129â 4139 (2017). www.fasebj.orgâ Fattahi, Fatemeh, Kalbitz, Miriam, Malan, Elizabeth A., Abe, Elizabeth, Jajou, Lawrence, Huberâ Lang, Markus S., Bosmann, Markus, Russell, Mark W., Zetoune, Firas S., Ward, Peter A., Complementâ induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction. FASEB J. 31, 4129â 4139 (2017)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154261/1/fsb2fj201700140r.pd