The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids

open5noBACKGROUND: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. METHODS: Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. RESULTS: The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. CONCLUSIONS: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. GENERAL SIGNIFICANCE: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis.openLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sara NLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sar

Granulin Knock Out Zebrafish Lack Frontotemporal Lobar Degeneration and Neuronal Ceroid Lipofuscinosis Pathology

Loss of function mutations in granulin (GRN) are linked to two distinct neurological disorders, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). It is so far unknown how a complete loss of GRN in NCL and partial loss of GRN in FTLD can result in such distinct diseases. In zebrafish, there are two GRN homologues, Granulin A (Grna) and Granulin B (Grnb). We have generated stable Grna and Grnb loss of function zebrafish mutants by zinc finger nuclease mediated genome editing. Surprisingly, the grna and grnb single and double mutants display neither spinal motor neuron axonopathies nor a reduced number of myogenic progenitor cells as previously reported for Grna and Grnb knock down embryos. Additionally, grna-/-;grnb-/- double mutants have no obvious FTLD- and NCL-related biochemical and neuropathological phenotypes. Taken together, the Grna and Grnb single and double knock out zebrafish lack any obvious morphological, pathological and biochemical phenotypes. Loss of zebrafish Grna and Grnb might therefore either be fully compensated or only become symptomatic upon additional challenge

Asynchronous 3D (Async3D): Design Methodology and Analysis of 3D Asynchronous Circuits

This dissertation focuses on the application of 3D integrated circuit (IC) technology on asynchronous logic paradigms, mainly NULL Convention Logic (NCL) and Multi-Threshold NCL (MTNCL). It presents the Async3D tool flow and library for NCL and MTNCL 3D ICs. It also analyzes NCL and MTNCL circuits in 3D IC. Several FIR filter designs were implement in NCL, MTNCL, and synchronous architecture to compare synchronous and asynchronous circuits in 2D and 3D ICs. The designs were normalized based on performance and several metrics were measured for comparison. Area, interconnect length, power consumption, and power density were compared among NCL, MTNCL, and synchronous designs. The NCL and MTNCL designs showed improvements in all metrics when moving from 2D to 3D. The 3D NCL and MTNCL designs also showed a balanced power distribution in post-layout analysis. This could alleviate the hotspot problem prevalently found in most 3D ICs. NCL and MTNCL have the potential to synergize well with 3D IC technology

Druggable Nucleolin Identifies Breast Tumours Associated with Poor Prognosis That Exhibit Different Biological Processes

Background: Nucleolin (NCL) is a multifunctional protein with oncogenic properties. Anti-NCL drugs show strong cytotoxic effects, including in triple-negative breast cancer (TNBC) models, and are currently being evaluated in phase II clinical trials. However, few studies have investigated the clinical value of NCL and whether NCL stratified cancer patients. Here, we have investigated for the first time the association of NCL with clinical characteristics in breast cancers independently of the different subtypes. Methods: Using two independent series (n = 216; n = 661), we evaluated the prognostic value of NCL in non-metastatic breast cancers using univariate and/or multivariate Cox-regression analyses. Results: We reported that NCL mRNA expression levels are markers of poor survivals independently of tumour size and lymph node invasion status (n = 216). In addition, an association of NCL expression levels with poor survival was observed in TNBC (n = 40, overall survival (OS) p = 0.0287, disease-free survival (DFS) p = 0.0194). Transcriptomic analyses issued from The Cancer Genome Atlas (TCGA) database (n = 661) revealed that breast tumours expressing either low or high NCL mRNA expression levels exhibit different gene expression profiles. These data suggest that tumours expressing high NCL mRNA levels are different from those expressing low NCL mRNA levels. Conclusions: NCL is an independent marker of prognosis in breast cancers. We anticipated that anti-NCL is a promising therapeutic strategy that could rapidly be evaluated in high NCL-expressing tumours to improve breast cancer management

Progressive retinal degeneration and glial activation in the Cln6nclf mouse model of neuronal ceroid lipofuscinosis : a beneficial effect of DHA and Curcumin supplementation

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds

Novel insights into the regulation of nucleolar function and longevity by ncl-1

The nucleolus is primarily described as the control center for ribonuclear protein assembly and rRNA synthesis. However, there is increasing evidence that the nucleolus also plays a role in protein quality, genome stability and cell cycle progression. Previous studies demonstrated that the B-box type zinc finger protein ncl- 1 is tightly linked to nucleolar function, and that its loss leads to increased levels of the nucleolar marker fibrillarin (fib-1) and to an increased nucleolar area. These nucleolar phenotypes are accompanied by the abolishment of lifespan extension in the major longevity pathways. However, the molecular connection between ncl-1, nucleolar function and longevity remains elusive. To identify molecular players and pathways that mediate the function of ncl-1 on longevity, I performed transcriptomic and proteomic analysis, comparing wild-type worms to ncl-1 worms in normal as well as the long-lived glp-1 background. This analysis revealed some shared and some background-specific regulation of distinct biological pathways upon loss of ncl-1: While nuclear outputs such as ribosome biogenesis and rRNA production are increased in multiple tested genotypes, proteasomal components are decreased in ncl-1 single mutants, while lysosomal components show lower abundance in glp-1;ncl-1. Interestingly, NCL-1 seems to affect at least a portion of regulated genes through direct binding of respective mRNAs. Based on transcriptomic and proteomic data, I conducted a follow-up RNA interference screen of potential mediators and uncovered nucleolar downstream processes of ncl- 1 including the RNase P/MRP complex and mitochondrial translation. Knockdown of the shared RNase P/MRP component POPL-1 as well as other RNase P/MRP factors extended lifespan in glp-1;ncl-1 mutants. The same was observed for other rRNA processing and ribosome assembly factors as well as proteins involved in mitochondrial translation such as MRPS-16. Reduction of popl-1 and mrps-16 extended lifespan independent of nucleolar size, fib-1 mRNA levels or steady state rRNA levels, thereby uncoupling nucleolar size from lifespan for the first time. Also, overall translation rate seems unaffected. Thus, the effect of NCL-1 on lifespan may be mediated through altered ribosome assembly in glp-1 worms, while being unaffected in the N2 background. To further investigate the molecular mechanism of NCL-1 action, I conducted a Yeast- 2-Hybrid assay and pulldown experiments to identify NCL-1 protein interactors. In a mini RNAi screen, I found that a reduction of the potential NCL-1 interactor and proteasomal factor RPN-11 affects nucleolar size in glp-1 mutants, indicating a direct connection of NCL-1 to the proteasome. In line with this, ncl-1 affects cellular proteostasis with the proteasome as a presumable key player, as ncl-1 mutants are short-lived under modest thermal stress and are less motile. In addition, I observed that overall ubiquitinylation levels are increased, and that proteasome substrates accumulate in ncl-1 mutants. In the pulldown experiments with NCL-1, I identified several mitochondrial factors in the co-enriched fraction of NCL-1. I also found that NCL-1 forms round network-like structures and strong foci resembling mitochondria within the hypodermis and muscle, further supporting a mitochondrial connection. Further on I established a connection between the RNAi machinery, nucleolar size and lifespan with a central focus on the argonaut protein NRDE-3 being required for longevity and affecting nucleolar size. Using a range of genetic and biochemical approaches, I found that ncl-1 is a multifaceted gene that connects multiple important cellular pathways with a focus on nucleolar outputs, but also proteolytic processes. I identified the RNase P/MRP complex and mitochondrial translation as potential key processes for mediating the function of ncl-1 in longevity

A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies

A murine model of variant late infantile ceroid lipofuscinosis recapitulates behavioral and pathological phenotypes of human disease.

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6(nclf)) mouse line to validate its utility for translational research. We demonstrate that this Cln6(nclf) mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6(nclf) mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics