Evaluating the miR-302b and miR-145 expression in formalin-fixed paraffin-embedded samples of esophageal squamous cell carcinoma

Background: MicroRNAs are involved in key cellular processes regulating, and their misregulation is linked to cancer. The miR-302-367 cluster is exclusively expressed in embryonic stem and carcinoma cells. This cluster also promotes cell reprogramming and stemness process. In contrast, miR-145 is mostly regarded as a tumor suppressor, where it regulates cellular functions such as cell division, differentiation, and apoptosis. By suppressing the main pluripotency factors (OCT4, SOX2, MYC and KLF4), miR-145 silences the self-renewal program in ESCs. Therefore, the main aim of this study is to find a potential link between the expression level of hsa-miR-302b and hsa-miR-145 with tumor vs. non-tumor as well as high-grade vs. low-grade states of the esophageal tissue samples. Methods: A total number of 40 formalin-fixed, paraffin-embedded (FFPE) samples of esophageal squamous-cell carcinoma (ESCC) were obtained, and the tumor and marginal non-tumor areas delineated and punched off by an expert pathologist. Total RNA was extracted with Trizol, and cDNA synthesized using the miRCURY LNA™ Universal RT microRNA PCR Kit. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were performed using specific LNA-primers and SYBR Green master mix. Results: The expression level of miR-302b failed to show any significant difference, neither between tumor and their non-tumor counterparts, nor among tumors with different grades of malignancies (P > 0.05). In contrast, miR-145 was significantly down regulated in all grades of tumor samples (P 0.05). CONCLUSION: Our data revealed a significant down-regulation of miR-145 in ESCC tissue samples. Based on our ROC curve analysis data (AUC = 0.74, P < 0.001) miR-145 could be regarded as a potential tumor marker for diagnosis of esophageal cancer. © 2015, Academy of Medical Sciences of I.R. Iran. All rights reserved

Interregional compensatory mechanisms of motor functioning in progressing preclinical neurodegeneration.

Understanding brain reserve in preclinical stages of neurodegenerative disorders allows determination of which brain regions contribute to normal functioning despite accelerated neuronal loss. Besides the recruitment of additional regions, a reorganisation and shift of relevance between normally engaged regions are a suggested key mechanism. Thus, network analysis methods seem critical for investigation of changes in directed causal interactions between such candidate brain regions. To identify core compensatory regions, fifteen preclinical patients carrying the genetic mutation leading to Huntington's disease and twelve controls underwent fMRI scanning. They accomplished an auditory paced finger sequence tapping task, which challenged cognitive as well as executive aspects of motor functioning by varying speed and complexity of movements. To investigate causal interactions among brain regions a single Dynamic Causal Model (DCM) was constructed and fitted to the data from each subject. The DCM parameters were analysed using statistical methods to assess group differences in connectivity, and the relationship between connectivity patterns and predicted years to clinical onset was assessed in gene carriers. In preclinical patients, we found indications for neural reserve mechanisms predominantly driven by bilateral dorsal premotor cortex, which increasingly activated superior parietal cortices the closer individuals were to estimated clinical onset. This compensatory mechanism was restricted to complex movements characterised by high cognitive demand. Additionally, we identified task-induced connectivity changes in both groups of subjects towards pre- and caudal supplementary motor areas, which were linked to either faster or more complex task conditions. Interestingly, coupling of dorsal premotor cortex and supplementary motor area was more negative in controls compared to gene mutation carriers. Furthermore, changes in the connectivity pattern of gene carriers allowed prediction of the years to estimated disease onset in individuals. Our study characterises the connectivity pattern of core cortical regions maintaining motor function in relation to varying task demand. We identified connections of bilateral dorsal premotor cortex as critical for compensation as well as task-dependent recruitment of pre- and caudal supplementary motor area. The latter finding nicely mirrors a previously published general linear model-based analysis of the same data. Such knowledge about disease specific inter-regional effective connectivity may help identify foci for interventions based on transcranial magnetic stimulation designed to stimulate functioning and also to predict their impact on other regions in motor-associated networks

Rapid cell-surface prion protein conversion revealed using a novel cell system

Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion

Altered Intracortical T-1-Weighted/T-2-Weighted Ratio Signal in Huntington's Disease

Neurological Motor Disorder

Cross-sectional and longitudinal voxel-based grey matter asymmetries in Huntington's disease

Huntington's disease (HD) is a progressive neurodegenerative disorder that can be genetically confirmed with certainty decades before clinical onset. This allows the investigation of functional and structural changes in HD many years prior to disease onset, which may reveal important mechanistic insights into brain function, structure and organization in general. While regional atrophy is present at early stages of HD, it is still unclear if both hemispheres are equally affected by neurodegeneration and how the extent of asymmetry affects domain-specific functional decline. Here, we used whole-brain voxel-based analysis to investigate cross-sectional and longitudinal hemispheric asymmetries in grey matter (GM) volume in 56 manifest HD (mHD), 83 pre-manifest HD (preHD), and 80 healthy controls (HC). Furthermore, a regression analysis was used to assess the relationship between neuroanatomical asymmetries and decline in motor and cognitive measures across the disease spectrum. The cross-sectional analysis showed striatal leftward-biased GM atrophy in mHD, but not in preHD, relative to HC. Longitudinally, no net 36-month change in GM asymmetries was found in any of the groups. In the regression analysis, HD-related decline in quantitative-motor (Q-Motor) performance was linked to lower GM volume in the left superior parietal cortex. These findings suggest a stronger disease effect targeting the left hemisphere, especially in those with declining motor performance. This effect did not change over a period of three years and may indicate a compensatory role of the right hemisphere in line with recent functional imaging studies

Apathy predicts rate of cognitive decline over 24 months in premanifest Huntington's disease

Background Cognitive impairment is a core feature of Huntington's disease (HD), however, the onset and rate of cognitive decline is highly variable. Apathy is the most common neuropsychiatric symptom of HD, and is associated with cognitive impairment. The aim of this study was to investigate apathy as a predictor of subsequent cognitive decline over 2 years in premanifest and early HD, using a prospective, longitudinal design. Methods A total of 118 premanifest HD gene carriers, 111 early HD and 118 healthy control participants from the multi-centre TRACK-HD study were included. Apathy symptoms were assessed at baseline using the apathy severity rating from the Short Problem Behaviours Assessment. A composite of 12 outcome measures from nine cognitive tasks was used to assess cognitive function at baseline and after 24 months. Results In the premanifest group, after controlling for age, depression and motor signs, more apathy symptoms predicted faster cognitive decline over 2 years. In contrast, in the early HD group, more motor signs, but not apathy, predicted faster subsequent cognitive decline. In the control group, only older age predicted cognitive decline. Conclusions Our findings indicate that in premanifest HD, apathy is a harbinger for cognitive decline. In contrast, after motor onset, in early diagnosed HD, motor symptom severity more strongly predicts the rate of cognitive decline.Neurological Motor Disorder

Validating automated segmentation tools in the assessment of caudate atrophy in Huntington's disease

Background: Neuroimaging shows considerable promise in generating sensitive and objective outcome measures for therapeutic trials across a range of neurodegenerative conditions. For volumetric measures the current gold standard is manual delineation, which is unfeasible for samples sizes required for large clinical trials.Methods: Using a cohort of early Huntington's disease (HD) patients (n = 46) and controls (n = 35), we compared the performance of four automated segmentation tools (FIRST, FreeSurfer, STEPS, MALP-EM) with manual delineation for generating cross-sectional caudate volume, a region known to be vulnerable in HD. We then examined the effect of each of these baseline regions on the ability to detect change over 15 months using the established longitudinal Caudate Boundary Shift Integral (cBSI) method, an automated longitudinal pipeline requiring a baseline caudate region as an input.Results: All tools, except Freesurfer, generated significantly smaller caudate volumes than the manually derived regions. Jaccard indices showed poorer levels of overlap between each automated segmentation and manual delineation in the HD patients compared with controls. Nevertheless, each method was able to demonstrate significant group differences in volume (p < 0.001). STEPS performed best qualitatively as well as quantitively in the baseline analysis. Caudate atrophy measures generated by the cBSI using automated baseline regions were largely consistent with those derived from a manually segmented baseline, with STEPS providing the most robust cBSI values across both control and HD groups.Conclusions: Atrophy measures from the cBSI were relatively robust to differences in baseline segmentation technique, suggesting that fully automated pipelines could be used to generate outcome measures for clinical trials.Neurological Motor Disorder

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for H

Testing a longitudinal compensation model in premanifest Huntington's disease

Neurological Motor Disorder

Subcellular localization and formation of huntingtin aggregates correlates with symptom onset and progression in a Huntington's disease model

Huntington's disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington's disease, that were known to abrogate somatic instability in Huntington's disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington's disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington's disease patient's blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)(90) mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG)(200) mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)(90) brains have different properties to those in the R6/2(CAG)(200) mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)(200) brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes