Broad clinical phenotypes associated with TAR-DNA binding protein (TARDBP) mutations in amyotrophic lateral sclerosis

The finding of TDP-43 as a major component of ubiquitinated protein inclusions in amyotrophic lateral sclerosis (ALS) has led to the identification of 30 mutations in the transactive response-DNA binding protein (TARDBP) gene, encoding TDP-43. All but one are in exon 6, which encodes the glycine-rich domain. The aim of this study was to determine the frequency of TARDBP mutations in a large cohort of motor neurone disease patients from Northern England (42 non-superoxide dismutase 1 (SOD1) familial ALS (FALS), nine ALS-frontotemporal dementia, 474 sporadic ALS (SALS), 45 progressive muscular atrophy cases). We identified four mutations, two of which were novel, in two familial (FALS) and two sporadic (SALS) cases, giving a frequency of TARDBP mutations in non-SOD1 FALS of 5% and SALS of 0.4%. Analysis of clinical data identified that patients had typical ALS, with limb or bulbar onset, and showed considerable variation in age of onset and rapidity of disease course. However, all cases had an absence of clinically overt cognitive dysfunction

Is there a space–time continuum in olfaction?

The coding of olfactory stimuli across a wide range of organisms may rely on fundamentally similar mechanisms in which a complement of specific odorant receptors on olfactory sensory neurons respond differentially to airborne chemicals to initiate the process by which specific odors are perceived. The question that we address in this review is the role of specific neurons in mediating this sensory system—an identity code—relative to the role that temporally specific responses across many neurons play in producing an olfactory perception—a temporal code. While information coded in specific neurons may be converted into a temporal code, it is also possible that temporal codes exist in the absence of response specificity for any particular neuron or subset of neurons. We review the data supporting these ideas, and we discuss the research perspectives that could help to reveal the mechanisms by which odorants become perceptions

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications

The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis

The discovery that a hexanucleotide repeat expansion in C9orf72 is the most numerous genetic variant of both amyotrophic lateral sclerosis and frontotemporal dementia has opened a rapidly growing field, which may provide long hoped for advances in the understanding and treatment of these devastating diseases. In this review we describe the various phenotypes, clinical and pathological, associated with expansion of C9orf72, which go beyond amyotrophic lateral sclerosis and frontotemporal dementia to include neurodegeneration more broadly. Next we take a step back and summarize the current understanding of the C9orf72 expansion and its protein products at a molecular level. Three mechanisms are prominent: toxicity mediated directly by RNA transcribed from the repeat; toxicity mediated by dipeptide repeat proteins translated from the repeat sequence; and haploinsufficiency resulting from reduced transcription of the C9orf72 exonic sequence. A series of exciting advances have recently described how dipeptide repeat proteins might interfere with the normal role of the nucleolus in maturation of RNA binding proteins and in production of ribosomes. Importantly, these mechanisms are unlikely to be mutually exclusive. We draw attention to the fact that clinical and pathological similarities to other genetic variants without a repeat expansion must not be overlooked in ascribing a pathogenic mechanism to C9orf72-disease. Finally, with a view to impact on patient care, we discuss current practice with respect to genetic screening in patients with and without a family history of disease, and the most promising developments towards therapy that have been reported to date