EVOLUTION OF THE CIRCADIAN CLOCK IN EXTREME ENVIRONMENT: LESSONS FROM CAVEFISH.

Evolution has been strongly influenced by the daily cycles of temperature and light imposed by the rotation of the Earth. Fascinating demonstrations of this are seen in extreme environments such as caves where some animals have remained completely isolated from the day-night cycle for millions of years. Most of these species show convergent evolution, sharing a range of striking physical properties such as eye loss. One fundamental issue is whether “hypogean” species retain a functional circadian clock. This highly conserved, physiological timing mechanism allows organisms to anticipate daily environmental changes and is synchronized primarily by light. The Somalian cavefish, Phreatichthys andruzzii does possess a circadian clock that is entrained by a daily regular feeding time but strikingly, not by light. Under constant conditions the P. andruzzii clock oscillates with an extremely long period and also lacks normal temperature compensation. We document multiple mutations affecting a light-induced clock gene, Period2 as well as the genes encoding the extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin. Remarkably, we show that ectopic expression of zebrafish homologs of these opsins rescues light induced clock gene expression in P. andruzzii cells. Thus, by studying this natural mutant we provide direct evidence for a peripheral light-sensing function of extra-retinal opsins in vertebrates. Furthermore, the properties of this cavefish illustrate that evolution in constant darkness leads not only to anatomical changes but also to loss of gene function linked with the detection and anticipation of the day-night cycle

Thermo-Sensitive Alternative Splicing of FLOWERING LOCUS M Is Modulated by Cyclin-Dependent Kinase G2

The ability to sense environmental temperature and to coordinate growth and development accordingly, is critical to the reproductive success of plants. Flowering time is regulated at the level of gene expression by a complex network of factors that integrate environmental and developmental cues. One of the main players, involved in modulating flowering time in response to changes in ambient temperature is FLOWERING LOCUS M (FLM). FLM transcripts can undergo extensive alternative splicing producing multiple variants, of which FLM-β and FLM-δ are the most representative. While FLM-β codes for the flowering repressor FLM protein, translation of FLM-δ has the opposite effect on flowering. Here we show that the cyclin-dependent kinase G2 (CDKG2), together with its cognate cyclin, CYCLYN L1 (CYCL1) affects the alternative splicing of FLM, balancing the levels of FLM-β and FLM-δ across the ambient temperature range. In the absence of the CDKG2/CYCL1 complex, FLM-β expression is reduced while FLM-δ is increased in a temperature dependent manner and these changes are associated with an early flowering phenotype in the cdkg2 mutant lines. In addition, we found that transcript variants retaining the full FLM intron 1 are sequestered in the cell nucleus. Strikingly, FLM intron 1 splicing is also regulated by CDKG2/CYCL1. Our results provide evidence that temperature and CDKs regulate the alternative splicing of FLM, contributing to flowering time definition

A Functional Kinase Is Necessary for Cyclin-Dependent Kinase G1 (CDKG1) to Maintain Fertility at High Ambient Temperature in Arabidopsis

Maintaining fertility in a fluctuating environment is key to the reproductive success of flowering plants. Meiosis and pollen formation are particularly sensitive to changes in growing conditions, especially temperature. We have previously identified cyclin-dependent kinase G1 (CDKG1) as a master regulator of temperature-dependent meiosis and this may involve the regulation of alternative splicing (AS), including of its own transcript. CDKG1 mRNA can undergo several AS events, potentially producing two protein variants: CDKG1L and CDKG1S, differing in their N-terminal domain which may be involved in co-factor interaction. In leaves, both isoforms have distinct temperature-dependent functions on target mRNA processing, but their role in pollen development is unknown. In the present study, we characterize the role of CDKG1L and CDKG1S in maintaining Arabidopsis fertility. We show that the long (L) form is necessary and sufficient to rescue the fertility defects of the cdkg1-1 mutant, while the short (S) form is unable to rescue fertility. On the other hand, an extra copy of CDKG1L reduces fertility. In addition, mutation of the ATP binding pocket of the kinase indicates that kinase activity is necessary for the function of CDKG1. Kinase mutants of CDKG1L and CDKG1S correctly localize to the cell nucleus and nucleus and cytoplasm, respectively, but are unable to rescue either the fertility or the splicing defects of the cdkg1-1 mutant. Furthermore, we show that there is partial functional overlap between CDKG1 and its paralog CDKG2 that could in part be explained by overlapping gene expression

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition

Mobility impairment is associated with reduced microstructural integrity of the inferior and superior cerebellar peduncles in elderly with no clinical signs of cerebellar dysfunction☆

While the cerebellum plays a critical role in motor coordination and control no studies have investigated its involvement in idiopathic mobility impairment in community-dwelling elderly. In this study we tested the hypothesis that structural changes in the cerebellar peduncles not detected by conventional magnetic resonance imaging are associated with reduced mobility performance. The analysis involved eighty-five subjects (age range: 75–90 years) who had no clinical signs of cerebellar dysfunction. Based on the short physical performance battery (SPPB) score, we defined mobility status of the subjects in the study as normal (score 11–12, n = 26), intermediate (score 9–10, n = 27) or impaired (score < 9, n = 32). We acquired diffusion tensor imaging data to obtain indices of white matter integrity: fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD). Using a parcellation atlas, regional indices within the superior, middle, and inferior cerebellar peduncles (ICP, MCP, SCP) were calculated and their associations with mobility performance were analyzed. Subjects with impaired mobility showed reduced FA and AD values in the ICP and SCP but not in the MCP. The ICP-FA, ICP-AD and SCP-FA indices showed a significant association with the SPPB score. We also observed significant correlation between ICP-FA and walk time (r = − 0.311, p = 0.004), as well as between SCP-AD and self-paced maximum walking velocity (r = 0.385, p = 0.003) and usual walking velocity (r = 0.400, p = 0.002). In logistic regression analysis ICP-FA and ICP-AD together explained 51% of the variability in the mobility status of a sample comprising the normal and impaired subgroups, and correctly classified more than three-quarters of those subjects. Our findings suggest that presence of microstructural damage, likely axonal, in afferent and efferent connections of the cerebellum contributes to the deterioration of motor performance in older people

Mutations in blind cavefish target the light-regulated circadian clock gene, period 2

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.Universidad de Ferrara | Ref. FAR2014-201

Chronic Obstructive Pulmonary Disease Is Associated with Altered Neuropsychological Performance in Young Adults

Subjects with ischemic lesions have an increased risk of dementia. In addition, Alzheimer's disease (AD) and vascular cognitive impairment share many risk factors. These observations suggest that different diseases that cause altered blood perfusion of the brain or hypoxia promote AD neurodegeneration. In this case-control, cross-sectional study, we sought to test the hypothesis that hypoxia facilitates cognitive decline. We looked for altered neuropsychological performance in subjects with chronic obstructive pulmonary disease (COPD) without apparent cardio- or cerebrovascular diseases or risk factors for atherosclerosis. A selected, homogeneous group of workers from two ceramic factories in a small town of central Italy was enrolled in this study. The COPD patients had a slightly, but significantly worse performance than controls in a number of neuropsychological tests. The findings are consistent with the working hypothesis that chronic hypoxia facilitates cognitive decline

Microstructural Changes in the Striatum and Their Impact on Motor and Neuropsychological Performance in Patients with Multiple Sclerosis

Grey matter (GM) damage is a clinically relevant feature of multiple sclerosis (MS) that has been previously assessed with diffusion tensor imaging (DTI). Fractional anisotropy (FA) of the basal ganglia and thalamus might be increased in MS patients, and correlates with disability scores. Despite the established role of the striatum and thalamus in motor control, mood and cognition, the impact of DTI changes within these structures on motor and neuropsychological performance has not yet been specifically addressed in MS. We investigated DTI metrics of deep GM nuclei and their potential association with mobility and neuropsychological function. DTI metrics from 3T MRI were assessed in the caudate, putamen, and thalamus of 30 MS patients and 10 controls. Sixteen of the patients underwent neuropsychological testing. FA of the caudate and putamen was higher in MS patients compared to controls. Caudate FA correlated with Expanded Disability Status Scale score, Ambulation Index, and severity of depressive symptomatology. Putamen and thalamus FA correlated with deficits in memory tests. In contrast, cerebral white matter (WM) lesion burden showed no significant correlation with any of the disability, mobility and psychometric parameters. Our findings support evidence of FA changes in the basal ganglia in MS patients, as well as deep GM involvement in disabling features of MS, including mobility and cognitive impairment. Deep GM FA appears to be a more sensitive correlate of disability than WM lesion burden

Targeting alternative splicing by RNAi: From the differential impact on splice variants to triggering artificial pre-mRNA splicing

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.Fil: Fuchs, Armin. Universidad de Viena; AustriaFil: Riegler, Stefan. Universidad de Viena; AustriaFil: Ayatollahi, Zahra. Universidad de Viena; AustriaFil: Cavallari, Nicola. Universidad de Viena; AustriaFil: Giono, Luciana Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nimeth, Barbara A.. University of Natural Resources and Life Sciences ; AustriaFil: Mutanwad, Krishna V.. University of Natural Resources and Life Sciences ; AustriaFil: Schweighofer, Alois. Universidad de Viena; AustriaFil: Lucyshyn, Doris. Universitat Fur Bodenkultur Wien; AustriaFil: Barta, Andrea. University of Natural Resources and Life Sciences ; AustriaFil: Petrillo, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Kalyna, Maria. University of Natural Resources and Life Sciences ; Austri

Dual‐Sensitivity Multiple Sclerosis Lesion and CSF Segmentation for Multichannel 3T Brain MRI

ABSTRACT BACKGROUND AND PURPOSE A pipeline for fully automated segmentation of 3T brain MRI scans in multiple sclerosis (MS) is presented. This 3T morphometry (3TM) pipeline provides indicators of MS disease progression from multichannel datasets with high‐resolution 3‐dimensional T1‐weighted, T2‐weighted, and fluid‐attenuated inversion‐recovery (FLAIR) contrast. 3TM segments white (WM) and gray matter (GM) and cerebrospinal fluid (CSF) to assess atrophy and provides WM lesion (WML) volume. METHODS To address nonuniform distribution of noise/contrast (eg, posterior fossa in 3D‐FLAIR) of 3T magnetic resonance imaging, the method employs dual sensitivity (different sensitivities for lesion detection in predefined regions). We tested this approach by assigning different sensitivities to supratentorial and infratentorial regions, and validated the segmentation for accuracy against manual delineation, and for precision in scan‐rescans. RESULTS Intraclass correlation coefficients of .95, .91, and .86 were observed for WML and CSF segmentation accuracy and brain parenchymal fraction (BPF). Dual sensitivity significantly reduced infratentorial false‐positive WMLs, affording increases in global sensitivity without decreasing specificity. Scan‐rescan yielded coefficients of variation (COVs) of 8% and .4% for WMLs and BPF and COVs of .8%, 1%, and 2% for GM, WM, and CSF volumes. WML volume difference/precision was .49 ± .72 mL over a range of 0–24 mL. Correlation between BPF and age was r = .62 (P = .0004), and effect size for detecting brain atrophy was Cohen's d = 1.26 (standardized mean difference vs. healthy controls). CONCLUSIONS This pipeline produces probability maps for brain lesions and tissue classes, facilitating expert review/correction and may provide high throughput, efficient characterization of MS in large datasets