Engineering synucleinopathy‐resistant human dopaminergic neurons by CRISPR‐mediated deletion of the SNCA gene

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA+/− and SNCA−/− cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA+/− and SNCA−/− neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology

Direct Data Distribution From Low-Earth Orbit

NASA Lewis Research Center (LeRC) is developing the space and ground segment technologies necessary to demonstrate a direct data distribution (1)3) system for use in space-to-ground communication links from spacecraft in low-Earth orbit (LEO) to strategically located tracking ground terminals. The key space segment technologies include a K-band (19 GHz) MMIC-based transmit phased array antenna, and a multichannel bandwidth- and power-efficient digital encoder/modulate with an aggregate data rate of 622 Mb/s. Along with small (1.8 meter), low-cost tracking terminals on the ground, the D3 system enables affordable distribution of data to the end user or archive facility through interoperability with commercial terrestrial telecommunications networks. The D3 system is applicable to both government and commercial science and communications spacecraft in LEO. The features and benefits of the D3 system concept are described. Starting with typical orbital characteristics, a set of baseline requirements for representative applications is developed, including requirements for onboard storage and tracking terminals, and sample link budgets are presented. Characteristics of the transmit array antenna and digital encoder/modulator are described. The architecture and components of the tracking terminal are described, including technologies for the next generation terminal. Candidate flights of opportunity for risk mitigation and space demonstration of the D3 features are identified

A rapid and sensitive assay for quantification of siRNA efficiency and specificity

RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling

Tectonic Controls on Gas Hydrate Distribution off SW Taiwan

The northern part of the South China Sea is characterized by widespread occurrence of bottom simulating reflectors (BSR) indicating the presence of marine gas hydrate. Because the area covers both a tectonically inactive passive margin and the termination of a subduction zone, the influence of tectonism on the dynamics of gas hydrate systems can be studied in this region. Geophysical data show that there are multiple thrust faults on the active margin while much fewer and smaller faults exist in the passive margin. This tectonic difference matches with a difference in the geophysical characteristics of the gas hydrate systems. High hydrate saturation derived from ocean bottom seismometer data and controlled source electromagnetic data and conspicuous high‐amplitude reflections in P‐Cable 3D seismic data above the BSR are found in the anticlinal ridges of the active margin. In contrast all geophysical evidence for the passive margin points to normal to low hydrate saturations. Geochemical analyses of gas samples collected at seep sites on the active margin show methane with heavy δ13C isotope composition, while gas collected at the passive margin shows light carbon isotope composition. Thus, we interpret the passive margin as a typical gas hydrate province fuelled by biogenic production of methane and the active margin gas hydrate system as a system that is fuelled not only by biogenic gas production but also by additional advection of thermogenic methane from the subduction system

α-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage α-Synuclein Aggregation

α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC₅₀ (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated

ERK2 Suppresses Self-Renewal Capacity of Embryonic Stem Cells, but Is Not Required for Multi-Lineage Commitment

Activation of the FGF-ERK pathway is necessary for naïve mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification

Differentiation-dependent association of phosphorylated extracellular signal-regulated kinase with the chromatin of osteoblast-related genes

The ERK/MAP kinase pathway is an important regulator of gene expression and differentiation in postmitotic cells. To understand how this pathway controls gene expression in bone, we examined the subnuclear localization of P-ERK in differentiating osteoblasts. Induction of differentiation was accompanied by increased ERK phosphorylation and expression of osteoblast-related genes, including osteocalcin ( Bglap2 ) and bone sialoprotein ( Ibsp ). Confocal immunofluorescence microscopy revealed that P-ERK colocalized with the RUNX2 transcription factor in the nuclei of differentiating cells. Interestingly, a portion of this nuclear P-ERK was directly bound to the proximal promoter regions of Bglap2 and Ibsp . Furthermore, the level of P-ERK binding to chromatin increased with differentiation, whereas RUNX2 binding remained relatively constant. The P-ERK-chromatin interaction was seen only in RUNX2-positive cells, required intact RUNX2-selective enhancer sequences, and was blocked with MAPK inhibition. These studies show for the first time that RUNX2 specifically targets P-ERK to the chromatin of osteoblast-related genes, where it may phosphorylate multiple substrates, including RUNX2, resulting in altered chromatin structure and gene expression. © 2010 American Society for Bone and Mineral ResearchPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64899/1/90705_ftp.pd

Sideroflexin 3 is an α-synuclein-dependent mitochondrial protein that regulates synaptic morphology.

α-Synuclein plays a central role in Parkinson's disease, where it contributes to the vulnerability of synapses to degeneration. However, the downstream mechanisms through which α-synuclein controls synaptic stability and degeneration are not fully understood. Here, comparative proteomics on synapses isolated from α-synuclein-/- mouse brain identified mitochondrial proteins as primary targets of α-synuclein, revealing 37 mitochondrial proteins not previously linked to α-synuclein or neurodegeneration pathways. Of these, sideroflexin 3 (SFXN3) was found to be a mitochondrial protein localized to the inner mitochondrial membrane. Loss of SFXN3 did not disturb mitochondrial electron transport chain function in mouse synapses, suggesting that its function in mitochondria is likely to be independent of canonical bioenergetic pathways. In contrast, experimental manipulation of SFXN3 levels disrupted synaptic morphology at the Drosophila neuromuscular junction. These results provide novel insights into α-synuclein-dependent pathways, highlighting an important influence on mitochondrial proteins at the synapse, including SFXN3. We also identify SFXN3 as a new mitochondrial protein capable of regulating synaptic morphology in vivo

Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript

Detection of low-level, lineage-specific transcription aids in the identification of lineage-primed populations of ES cells provides a new framework for pluripotency