Biochemical and cellular analysis of human variants of the DYT1 dystonia protein, TorsinA/TOR1A.

Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA(+) protein family that localizes to the endoplasmic reticulum lumen and nuclear envelope. This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients: R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia.

Stearoyl CoA Desaturase Is Essential for Regulation of Endoplasmic Reticulum Homeostasis and Tumor Growth in Glioblastoma Cancer Stem Cells.

Inherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%-100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma.

Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models.

Background: Molecular profile of glioblastoma multiforme (GBM) revealed 4 subtypes, 2 of which, proneural and mesenchymal, have been predominantly observed, with the latter displaying a more aggressive phenotype and increased therapeutic resistance. Single-cell RNA sequencing revealed that multiple subtypes actually reside within the same tumor, suggesting cellular heterogeneity in GBM. Further, plasticity between these 2 subtypes is observed during tumor recurrence and in response to radiation therapy. Methods: Patient-derived GBM stemlike cells were cultured as neurospheres. These cells were differentiated in serum by attaching to the culture dishes. The "floating" cells that were not attached/differentiated were harvested from the conditioned medium. The characteristics of these cells were studied with limiting dilution assays and immunofluorescence staining. Cell growth and nuclear factor-kappaB (NFkB) activation were monitored using bioluminescent assays as well as quantitative polymerase chain reaction and western blotting. In vivo tumorigenesis was evaluated in orthotopic xenograft models using bioluminescence imaging. Results: Patient-derived GBM stemlike cells undergo differentiation by attaching to the culture dish in serum-containing medium. We observed that a small subset of these cells escape this adhesion/differentiation and grow as floating cells. These cells displayed enhanced cancer stem cell properties with a molecular and phenotypic mesenchymal signature, including resistance to radiation and targeted therapies, a more aggressive tumor formation, and NFkB activation. Conclusion: Our results endorse inherent intratumor molecular subtype heterogeneity in glioblastoma and provide a valuable approach to study phenotypic plasticity, which could be applied to find novel therapeutic strategies to eradicate this aggressive tumor and can be extended to other cancer types.

New methods for investigation of neuronal migration in embryonic brain explants.

BACKGROUND: Proper migration of neurons is essential for the formation and normal functioning of the nervous system. Defects in neuronal migration underlie a number of neurologic diseases in humans. Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration remain to be elucidated fully. Newborn neurons from the embryonic medial ganglionic eminence (MGE) migrate a long distance dorsally in the developing brain, giving rise to several types of interneurons in the neocortex. NEW METHOD: In this study, we developed an immunocytochemistry (ICC) protocol to stain neurons migrating out of the MGE explant embedded in Matrigel. We also established a protocol to efficiently transfect cells in MGE explants, achieving a transduction efficiency of more than 30%. COMPARISON WITH EXISTING METHOD: In addition, we developed microfluidic chambers for explants that allow visualization of the vectorial migration of individual neurons from mouse embryonic MGE explants. Our microfluidic system allows monitoring of the distribution of cellular organelles (e.g. Golgi) within migrating neurons which have been stained with commercial molecular dyes or transfected with adeno-associated virus (AAV) expressing reporter proteins. CONCLUSION: These methods provide new paradigms to study neuronal migration in real-time.

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia.

BACKGROUND: Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. NEW METHOD: The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. RESULTS: We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. COMPARISON WITH EXISTING METHOD: The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. CONCLUSION: We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment.