ASO targeting RBM3 temperature-controlled poison exon splicing prevents neurodegeneration in vivo.

Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease-modifying treatments are currently available. Increasing the expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold-induced expression. Genetic removal or antisense oligonucleotide (ASO)-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increased RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease-associated prion protein. Our promising results in mice support the possibility that RBM3-inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

Immunity-related GTPase induces lipophagy to prevent excess hepatic lipid accumulation.

BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks.

Age-Dependent Changes in the Pulmonary Renin-Angiotensin System Are Associated With Severity of Lung Injury in a Model of Acute Lung Injury in Rats.

OBJECTIVES: A growing body of evidence suggests that age affects the main pathophysiologic mechanisms of the acute respiratory distress syndrome. This may imply the need for developing age-tailored therapies for acute respiratory distress syndrome. However, underlying molecular mechanisms governing age-related susceptibility first need to be unraveled. In a rat model of acute lung injury, we investigated whether age affects the balance between the two key enzymes of the pulmonary renin-angiotensin system, angiotensin-converting enzyme, and angiotensin-converting enzyme 2. We hypothesized that aging shifts the balance toward the lung injury-promoting angiotensin-converting enzyme, which may form an explanation for the differences in severity of lung injury between different age groups. DESIGN: Prospective, randomized controlled animal study. SETTING: University medical research laboratory. SUBJECTS: Infant (15 ± 2 d), juvenile (37 ± 2 d), adult (4 ± 0.2 mo), and elderly (19.5 ± 0.5 mo) male RCCHan Wistar rats. INTERVENTIONS: Lung injury was induced by intratracheal administration of lipopolysaccharide (5 mg/kg) and 4 hours of mechanical ventilation (15 mL/kg). MEASUREMENTS AND MAIN RESULTS: In lipopolysaccharide-exposed and mechanical ventilated rats, angiotensin-converting enzyme activity in bronchoalveolar lavage fluid increased 3.2-fold in elderly when compared with infants. No changes in bronchoalveolar lavage fluid angiotensin-converting enzyme 2 activity were found. In addition, membrane-bound angiotensin-converting enzyme activity decreased. Together with the presence of angiotensin-converting enzyme-sheddase ADAM9 (a disintegrin and metalloproteinase domain-containing protein 9) and an age-dependent increase in tumor necrosis factor-α, an activator of ADAM9, these results indicate increased shedding of angiotensin-converting enzyme in the alveolar compartment, thereby shifting the balance toward the injurious pathway. This imbalance was associated with an increased inflammatory mediator response and more lung injury (wet-to-dry ratio and histology) in elderly rats. CONCLUSIONS: Increasing age is associated with an imbalance of the pulmonary renin-angiotensin system, which correlates with aggravated inflammation and more lung injury. These changes might form the ground for new therapeutic strategies in terms of dosing and effectiveness of renin-angiotensin system-modulating agents for treatment of acute respiratory distress syndrome.

Hemodynamic Effects of the Non-Peptidic Angiotensin-(1-7) Agonist AVE0991 in Liver Cirrhosis.

BACKGROUND & AIMS: Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1-7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1-7) agonist, AVE0991, in experimental cirrhosis. METHODS: Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991. RESULTS: In vivo, AVE0991 decreased portal pressure (PP) in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways (ROCK, p-Moesin). Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats. CONCLUSIONS: The non-peptidic agonist of Ang-(1-7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure.