New friends for Ago2 in neuronal plasticity

MicroRNAs have emerged as central regulators of cellular homeostasis and increasing evidence suggests that they play a key role in neuronal plasticity. Major efforts are made to define microRNA networks and their targets in the brain. The mechanisms by which microRNA activity is regulated are, however, relatively unexplored. In this issue of The EMBO Journal, Störchel et al ( 2015 ) screened for proteins that affect microRNA function in neurons. They identify Nova1 and Ncoa3 as novel regulators of miRNA activity and demonstrate that both proteins are essential for neuronal plasticity in a microRNA‐dependent manner.     

Thymic involution,a co‐morbidity factor in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating disease, characterized by extremely rapid loss of motor neurons. Our studies over the last decade have established CD4⁺ T cells as important players in central nervous system maintenance and repair. Those results, together with recent findings that CD4⁺ T cells play a protective role in mouse models of ALS, led us to the current hypothesis that in ALS, a rapid T‐cell malfunction may develop in parallel to the motor neuron dysfunction. Here, we tested this hypothesis by assessing thymic function, which serves as a measure of peripheral T‐cell availability, in an animal model of ALS (mSOD1 [superoxide dismutase] mice; G93A) and in human patients. We found a significant reduction in thymic progenitor‐cell content, and abnormal thymic histology in 3–4‐month‐old mSOD1 mice. In ALS patients, we found a decline in thymic output, manifested in the reduction in blood levels of T‐cell receptor rearrangement excision circles, a non‐invasive measure of thymic function, and demonstrated a restricted T‐cell repertoire. The morbidity of the peripheral immune cells was also manifested in the increase of pro‐apoptotic BAX/BCXL2 expression ratio in peripheral blood mononuclear cells (PBMCs) of these patients. In addition, gene expression screening in the same PBMCs, revealed in the ALS patients a reduction in key genes known to be associated with T‐cell activity, including: CD80, CD86, IFNG and IL18. In light of the reported beneficial role of T cells in animal models of ALS, the present observation of thymic dysfunction, both in human patients and in an animal model, might be a co‐pathological factor in ALS, regardless of the disease aetiology. These findings may lead to the development of novel therapeutic approaches directed at overcoming the thymic defect and T‐cell deficiency.     

Age‐associated microRNA expression in human peripheral blood is associated with all‐cause mortality and age‐related traits

Recent studies provide evidence of correlations of DNA methylation and expression of protein‐coding genes with human aging. The relations of microRNA expression with age and age‐related clinical outcomes have not been characterized thoroughly. We explored associations of age with whole‐blood microRNA expression in 5221 adults and identified 127 microRNAs that were differentially expressed by age at P < 3.3 × 10^?4 (Bonferroni‐corrected). Most microRNAs were underexpressed in older individuals. Integrative analysis of microRNA and mRNA expression revealed changes in age‐associated mRNA expression possibly driven by age‐associated microRNAs in pathways that involve RNA processing, translation, and immune function. We fitted a linear model to predict ‘microRNA age’ that incorporated expression levels of 80 microRNAs. MicroRNA age correlated modestly with predicted age from DNA methylation (r = 0.3) and mRNA expression (r = 0.2), suggesting that microRNA age may complement mRNA and epigenetic age prediction models. We used the difference between microRNA age and chronological age as a biomarker of accelerated aging (Δage) and found that Δage was associated with all‐cause mortality (hazards ratio 1.1 per year difference, P = 4.2 × 10^?5 adjusted for sex and chronological age). Additionally, Δage was associated with coronary heart disease, hypertension, blood pressure, and glucose levels. In conclusion, we constructed a microRNA age prediction model based on whole‐blood microRNA expression profiling. Age‐associated microRNAs and their targets have potential utility to detect accelerated aging and to predict risks for age‐related diseases.     

Lost & found: C9ORF72 and the autophagy pathway in ALS/FTD

C9ORF72 expression is reduced in a substantial number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which may contribute to disease pathogenesis. However, its normal molecular function remains unknown. In this issue of The EMBO Journal, Sellier et al ( 2016 ) identified a novel protein complex consisting of C9ORF72, WDR41, and SMCR8 that acts as a GDP‐GTP exchange factor (GEF) for RAB8a and RAB39b and is regulated by TBK1, whose partial loss of function also causes ALS and FTD. They further reveal a potential modulatory role for this novel complex in macroautophagy (autophagy), especially in the context of ataxin‐2 toxicity.     

Unmasking the skiptic task of TDP‐43

The mechanism by which mutations in TAR DNA‐binding protein 43 (TDP‐43) cause neurodegeneration remains incompletely understood. In this issue of The EMBO Journal, Fratta et al ( 2018 ) describe how a point mutation in the C‐terminal low complexity domain of TDP‐43 leads to the skipping of otherwise constitutively conserved exons. In vivo, this mutation triggers late‐onset progressive neuromuscular disturbances, as seen in amyotrophic lateral sclerosis (ALS), suggesting that TDP‐43 splicing gain‐of‐function contributes to ALS pathogenesis.     

Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMl 1640 (RPMI) at 37°C and 5% CO₂ causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO₂ and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resuming in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiaeα-agglutinin, encoded by AGα1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for a gglutinin-l ike s equence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AGα1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AGb1. These repeats are also present on the nucleotide level as a highly conserved 108bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and. In some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.     

Monitoring <Emphasis Type="Italic">ALS1</Emphasis> and <Emphasis Type="Italic">ALS3</Emphasis> Gene Expression During In Vitro <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation Under Continuous Flow Conditions

ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear.     

室内饲养与野外采集的松墨天牛幼虫microRNA表达谱比较分析

【目的】昆虫随着生长环境的变化常常会有不同的生物学特性,其表观遗传调控机制研究值得关注。松墨天牛是松树萎焉病中松材线虫的媒介昆虫,但松墨天牛在实验室饲养和野外的不同条件下,其形态及发育速率有较大的区别,其表观遗传响应机制并不明确。通过比较分析microRNA表达谱揭示室内饲养和野外采集松墨天牛幼虫之间的差异,以期为松墨天牛幼虫的表观遗传研究提供参考。【方法】使用illumina Hi Seq 2000平台进行microRNA高通量测序,得到了实验室饲养和野外采集松墨天牛老熟幼虫的表皮、中肠microRNA库。鉴定保守microRNA和预测新microRNA,并对microRNA进行差异表达分析、靶基因预测、靶基因GO注释和KEGG功能富集分析。【结果】在室内饲养的松墨天牛表皮、中肠中分别鉴定出16、14个microRNA;在野外生存的天牛表皮、中肠中均鉴定出13个microRNA。与表皮相比,中肠的miRNA的表达量更高。与野外采集相比,在室内饲养天牛的microRNA表达量更高。17个microRNA表达量在室内饲养与野外采集的天牛之间有显著差异,比如novel-mir-62127、novel-mir-184731、novel-mir-290819等有明显上调,novel-mir-251851等明显下调。差异表达的miRNA的靶基因的功能主要富集在氨基糖代谢、几丁质代谢等糖代谢和甘油磷脂代谢、脂肪酸代谢等脂代谢过程。【结论】室内饲养和野外采集松墨天牛老熟幼虫的microRNA库存在明显差异,且不同的组织microRNA表达谱存在明显差异,提示经历室内恒定培养条件的松墨天牛具有表观遗传调控特征,为进一步研究松墨天牛发育、代谢的microRNA调控机制奠定了基础。     

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.     

Airborne laser scanning for vegetation structure quantification in a south east Australian scrubby forest‐woodland

Airborne laser scanning (ALS) has the potential to capture a range vegetation structural metrics, but most studies have focussed on conifer or mixed conifer‐deciduous cool‐temperate or boreal forests. This study focuses on a warm‐temperate eucalypt forest, where two epochs of ALS data, captured approximately 2 years apart, were compared with plot and transect field data collected after the second ALS epoch. Linear regression was used to compare metrics from field and ALS data, and Student's t‐tests were used to compare metrics from the two ALS epochs. Statistically significant relationships were found for tree height (R² = 0.915; SE = 2.08 m; P < 0.01) and canopy cover (R² = 0.508; SE = 16.4%; P < 0.01). Foliage projective cover was also significantly correlated (R² = 0.916; SE = 4.5%; P < 0.01) at a 10‐m stratification, but not at the typically computed 2‐m stratification, because of the presence of a tall scrubby understorey. Statistically significant values were also obtained from ALS data captured 2 years earlier, although correlation was not as strong, most likely because of the greater interval between fieldwork and ALS capture. Importantly, significant agreement was found for all metrics when the two ALS epochs were compared, suggesting that the metrics are robust.     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

Semi‐dominant mutation in the cysteine‐rich receptor‐like kinase gene,ALS1, conducts constitutive defence response in rice

• Plants have evolved a sophisticated two‐branch defence system to prevent the growth and spread of pathogen infection. The novel Cys‐rich repeat (CRR) containing receptor‐like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi‐dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath.
• A map‐based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification.
• Most pathogenesis‐related (PR) genes and other defence‐related genes were activated and up‐regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed.
• The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA‐, JA‐ and NH1‐mediated defence responses in rice.

     

Hypercapnia is a Possible Determinant of the Function of the Blood-Cerebrospinal Fluid Barrier in Amyotrophic Lateral Sclerosis

Elevated cerebrospinal fluid (CSF)/serum quotients of albumin (Q_Alb) may occur in motor neuron diseases (MND) including amyotrophic lateral sclerosis (ALS), but the pathophysiologic mechanisms underlying these alterations are unclear. Evidence from animal experiments suggests that the arterial carbon dioxide level might affect the Q_Alb, i.e. the function of the blood-CSF barrier (BCB). We therefore compared basic CSF parameters in different forms of MND (ALS, n = 105; lower motor neuron diseases, n = 12; and upper motor neuron diseases, n = 7) and investigated the relationship between elevated Q_Alb and the arterial partial pressure of carbon dioxide (pCO₂) in ALS where respiratory insufficiency leads to hypercapnia in the course of the disease. Pathologic elevations of Q_Alb occurred in 32 of 124 MND patients. In ALS, Q_Alb significantly correlated with the arterial pCO₂ (r = 0.454; P = 0.001; n = 45). These data indicate that BCB dysfunction is a frequent finding in different forms of MND and may reflect distinct pathophysiological mechanisms. In ALS, an important underlying mechanism might be the influence of the arterial pCO₂ which may alter the CSF flow.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.     

内含子源性microRNA对内皮型一氧化氮合酶表达及血管内皮细胞增殖的作用

前期工作表明,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)第4内含子中的27碱基 (nucleotide,nt)重复序列是27-nt microRNA的来源,并对eNOS具有重要的调节作用．为进一步探讨该内含子源性27-nt microRNA参与调节eNOS表达的分子机制及其在内皮细胞增殖中的可能作用,通过构建27-nt microRNA高表达质粒,用脂质体将该质粒转染人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC),Western blot和RT-PCR检测该细胞系中eNOS蛋白和mRNA表达情况以及胞核转录因子的表达改变,并观察HUVEC增殖的变化情况．结果发现：27-nt microRNA 高表达能降低eNOSmRNA的水平和蛋白质表达;同时对转录因子Sp1、Ap1的蛋白质表达也产生了不同程度的抑制作用;转染后细胞的生长速度比未转染的细胞明显减慢,尤其转染了27-nt microRNA的双倍长度突变体(pEGP-mut-54nt-mi)质粒的HUVEC,其生长倍增时间比正常对照组明显延长达49.4%．结果表明,27-nt microRNA明显抑制eNOS蛋白及其mRNA表达,同时 HUVEC增殖受到明显抑制,转录因子Sp1 和Ap1 在27-nt microRNA对eNOS的表达调节中起重要作用．实验提示,内含子源性microRNA与转录因子共同参与对内皮细胞增殖及其相关性基因的表达调节,可能是众多真核细胞中某些疾病相关性基因表达自我调节的重要机制之一．     

Increased oxidative stress in patients with amyotrophic lateral sclerosis and the effect of edaravone administration

Objectives and methods: Compared to age-matched healthy controls (n?=?55), patients with amyotrophic lateral sclerosis (ALS) (n?=?26) showed increased oxidative stress as indicated by a significantly increased percentage of oxidized coenzyme Q10 (%CoQ10) in total plasma coenzyme Q10, a significantly decreased level of plasma uric acid, and a significantly decreased percentage of polyunsaturated fatty acids in total plasma free fatty acids (FFA). Therefore, the efficacy of edaravone, a radical scavenger, in these ALS patients was examined.

Results and discussion: Among 26 ALS patients, 17 received edaravone (30?mg/day, one to four times a week) for at least 3 months, and 13 continued for 6 months. Changes in revised ALS functional rating scale (ALSFRS-R) were significantly smaller in these patients than in edaravone-untreated ALS patients (n?=?19). Edaravone administration significantly reduced excursions of more than one standard deviation from the mean for plasma FFA levels and the contents of palmitoleic and oleic acids, plasma markers of tissue oxidative damage, in the satisfactory progress group (ΔALSFRS-R?≥?0) as compared to the ingravescent group (ΔALSFRS-R?<??5). Edaravone treatment increased plasma uric acid, suggesting that it is an effective scavenger of peroxynitrite. However, edaravone administration did not decrease %CoQ10. Therefore, combined treatment with agents such as coenzyme Q10 may further reduce oxidative stress in ALS patients.     

Is cerebrospinal fluid obtained for diagnostic purpose a good material for biomarker studies in amyotrophic lateral sclerosis?

Abstract

The cerebrospinal fluid (CSF) used for identification of molecular biomarkers in amyotrophic lateral sclerosis (ALS) is mainly obtained from lumbar puncture (LP) performed to exclude other causes of motor neuron damage.

Aim: The aim of the study was to analyze whether CSF of ALS patients obtained for diagnostic purposes is suitable for biomarker studies in the entire ALS population.

Material and methods: We analyzed the medical data, LP frequency and CSF parameters in 568 ALS patients.

Results: LP was performed in 34% of cases. Patients who underwent LP were significantly younger and more frequently presented limb onset ALS, there were no differences in the clinical phenotypes.

Conclusion: CSF obtained for diagnostic purposes can be used for biomarkers studies in ALS.     

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri : phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene. Received: 27 November 1998 / Accepted: 21 April 1999