Tissue-dependent Mechanism Research Articles

Abstract 14446: Bcl-x Short Isoform is Essential for Maintaining Multiple-Tissue Homeostasis

Regulated cell death is an essential piece of normal development and maintenance of tissue homeostasis, wherein BCL2 like 1 (BCL2L1) protein is involved in key signaling pathways. Alternative splicing in exon 2 of Bcl2l1 produces Bcl-x short (Bcl-xS) isoform. However, mutations in around 5’ splice site are classified as variants of uncertain significance in humans, and the isoform-specific role of endogenous Bcl-xS remains largely unexplored. Here we show the role of Bcl-xS in vivo by generating the mice with a mutation in 5’ splice site of the Bcl2l1 that inhibits Bcl-xS alternative splicing. Although loss of BCL2L1 leads to embryonic lethal with massive cell death, Bcl-xS knockout (KO) mice were born at a mendelian ratio and showed no overt abnormality until 3 months of age. Thereafter, the KO mice developed cardiac hypertrophy (heart weight/tibia length, 8.59+/-0.308 vs 11.61+/-0.631, p = 0.0005 and 2.13-fold increase in individual cardiomyocyte size, p = 0.0001) with contractile dysfunction (EF, 69.25+/-1.31 vs 56.57+/-3.07, p = 0.0028) and splenomegaly (spleen weight/tibia length, 5.32+/-0.25 vs 7.12+/-0.49, p = 0.0051) at 6 months of age. Despite that overexpression of Bcl-xS induced cell death in cardiomyocytes (2.497-fold compared to LacZ, p < 0.0001), there was unexpectedly no overt difference in the level of apoptosis in the KO heart (TUNEL, 0.033+/-0.003 vs 0.030+/-0.002 %, p = 0.359), but fibrosis was significantly increased in the KO heart (1.602+/-0.095 vs 2.340+/-0.145 %, p = 0.0017). Diabetes was not evident in the KO mice (blood glucose, 169.8+/-10.9 vs 153.3+/-8.46 mg/dl, p = 0.2595). Mechanistically, we found that the Akt/mTOR and JNK/cJun signaling are activated in the KO heart and the JNK/cJun signaling is activated with increased Bax expression in the KO spleen. These results suggest that Bcl-xS may be dispensable for development, but is essential for maintaining homeostasis of multiple organs, especially cardiosplenic network, in a tissue-dependent mechanism.

Broadening the phenotypic spectrum and physiological insights related to EIF2S3 variants.

Mental deficiency, epilepsy, hypogonadism, microcephaly, and obesity syndrome is a severe X-linked syndrome caused by pathogenic variants in EIF2S3. The gene encodes the γ subunit of the eukaryotic translation initiation factor-2, eIF2, essential for protein translation. A recurrent frameshift variant is described in severely affected patients while missense variants usually cause a moderate phenotype. We identified a novel missense variant (c.433A>G, p.(Met145Val)) in EIF2S3 in a mildly affected patient. Studies on zebrafish confirm the pathogenicity of this novel variant and three previously published missense variants. CRISPR/Cas9 knockout of eif2s3 in zebrafish embryos recapitulate the human microcephaly and show increased neuronal cell death. Abnormal high glucose levels were identified in mutant embryos, caused by beta cell and pancreatic progenitor deficiency, not related to apoptosis. Additional studies in patient-derived fibroblasts did not reveal apoptosis. Our results provide new insights into disease physiopathology, suggesting tissue-dependent mechanisms.

A potential role of fatty acid binding protein 4 in the pathophysiology of autism spectrum disorder.

Autism spectrum disorder is a neurodevelopmental disorder characterized by difficulties in social communication and interaction, as well as repetitive and characteristic patterns of behaviour. Although the pathogenesis of autism spectrum disorder is unknown, being overweight or obesity during infancy and low weight at birth are known as risks, suggesting a metabolic aspect. In this study, we investigated adipose tissue development as a pathophysiological factor of autism spectrum disorder by examining the serum levels of adipokines and other metabolic markers in autism spectrum disorder children (n = 123) and typically developing children (n = 92) at 4–12 years of age. Among multiple measures exhibiting age-dependent trajectories, the leptin levels displayed different trajectory patterns between autism spectrum disorder and typically developing children, supporting an adipose tissue-dependent mechanism of autism spectrum disorder. Of particular interest, the levels of fatty acid binding protein 4 (FABP4) were significantly lower in autism spectrum disorder children than in typically developing subjects, at preschool age (4–6 years old: n = 21 for autism spectrum disorder and n = 26 for typically developing). The receiver operating characteristic curve analysis discriminated autism spectrum disorder children from typically developing children with a sensitivity of 94.4% and a specificity of 75.0%. We re-sequenced the exons of the FABP4 gene in a Japanese cohort comprising 659 autism spectrum disorder and 1000 control samples, and identified two rare functional variants in the autism spectrum disorder group. The Trp98Stop, one of the two variants, was transmitted to the proband from his mother with a history of depression. The disruption of the Fabp4 gene in mice evoked autism spectrum disorder-like behavioural phenotypes and increased spine density on apical dendrites of pyramidal neurons, which has been observed in the postmortem brains of autism spectrum disorder subjects. The Fabp4 knockout mice had an altered fatty acid composition in the cortex. Collectively, these results suggest that an ‘adipo-brain axis’ may underlie the pathophysiology of autism spectrum disorder, with FABP4 as a potential molecule for use as a biomarker.

Adipocyte JAK2 mediates growth hormone-induced hepatic insulin resistance.

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue-dependent mechanism.

Class III beta-tubulin, drug resistance and therapeutic approaches in cancers

Class III beta-tubulin is one of the critical proteins associated with microtubule assembly, important to many cellular functions including mitochondrial respiration and intracellular trafficking. Widely regarded as a specific neuronal marker in developmental neurobiology and stem cell research, it is also highly expressed in a wide range of tumors of both neuronal and non-neuronal origin. The expression of class III beta-tubulin is tightly controlled at multiple levels with tissue-dependent mechanisms of regulation. For instance, class III beta-tubulin expression is under the control of estrogens in breast cancer cells but is influenced by exposure to hypoxia and poor-nutrient supply in ovarian cancer. In some but not all cancers, class III beta-tubulin expression is purely a prognostic biomarker, predicting poor outcome of patients regardless of chemotherapy treatment. Moreover, the expression of class III beta-tubulin does not confer an aggressive phenotype by itself. Instead, class III beta-tubulin functions like a cytoskeletal gateway, which enhances the incorporation of pro-survival kinases into the cytoskeleton and protects them from degradation. The associations of class III beta-tubulin with survival kinase PIM-1, RNA-binding protein HuR, microRNAs are examples highlighting the functional complexity of this protein. The utility of class III beta-tubulin as a prognostic biomarker can also greatly improve if combined with these pro-survival partners. Subsequently, pharmacogenetic approaches, designed to counteract and target these pathways and associated-factors concurrently, might lead to better therapies and prognostic tools for class III beta-tubulin expressing cancers.

Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging.

BackgroundComplex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease.ResultsGene-targeting was used to delete exon 10 in murine tau on both alleles (E10−/−) to study its functional role. Moreover, mice devoid of exon 10 (E10+/−) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/− and wild-type mice, and their muscular grip strength was less than that of E10+/− mice. The performance of E10+/− mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.ConclusionAblation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions.

Complicated biallelic inactivation of Pten in radiation-induced mouse thymic lymphomas

Inactivation of the phosphatase and tensin homolog gene (Pten) occurs via multiple tissue-dependent mechanisms including epigenetic silencing, point mutations, insertions, and deletions. Although frequent loss of heterozygosity around the Pten locus and plausible involvement of epigenetic silencing have been reported in radiation-induced thymic lymphomas, the proportion of lymphomas with inactivated Pten and the spectrum of causal aberrations have not been extensively characterized. Here, we assessed the mode of Pten inactivation by comprehensive analysis of the expression and alteration of Pten in 23 radiation-induced thymic lymphomas developed in B6C3F1 mice. We found no evidence for methylation-associated silencing of Pten; rather, complex structural abnormalities comprised of missense and nonsense mutations, 1- and 3-bp insertions, and focal deletions were identified in 8 of 23 lymphomas (35%). Sequencing of deletion breakpoints suggested that aberrant V(D)J recombination and microhomology-mediated rearrangement were responsible for the focal deletions. Seven of the 8 lymphomas had biallelic alterations, and 4 of them did not express Pten protein. These Pten aberrations coincided with downstream Akt phosphorylation. In conclusion, we demonstrate that Pten inactivation is frequently biallelic and is caused by a variety of structural abnormalities (rather than by epigenetic silencing) and is involved in radiation-induced lymphomagenesis.

Tissue-Specific expression of rat kininogen mRNAs.

To characterize the local kallikrein-kinin system, tissue distribution of mRNAs for kininogens, precursor proteins of kinins, such as high-molecular-weight (H-), low-molecular-weight (L-) and T-kininogens, were studied in the rat by means of reverse-transcription polymerase chain reaction (RT-PCR) using a fluorophore Cy5-labeled 5'-primer. High levels of these three kininogen mRNAs were present in the liver. Relatively high levels of H-kininogen mRNA were also detected in the skin, lung, kidney, and testis in a descending order, whereas L-kininogen mRNA was detectable in the lung and brain, but not in the kidney, skin, testis, heart, adrenal gland, or skeletal muscle. T-Kininogen mRNA was present in these tissues, except for skeletal muscle. These findings suggest that the expression of each kininogen mRNA is regulated by the tissue-dependent mechanisms which is closely associated with functions of the kallikrein-kinin system in the tissue.