ABSTRACT
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.
Subject(s)
Cell Movement/physiology , Median Eminence/cytology , Neurons/physiology , Animals , Antigens/metabolism , Dependovirus/genetics , Embryo, Mammalian , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Microfluidic Analytical Techniques , Organ Culture Techniques , Transduction, Genetic , Tubulin/metabolismABSTRACT
TorsinA is an AAA+ ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508) was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VIMP and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing CFTRΔF508, and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state.
Subject(s)
Dystonia Musculorum Deformans/physiopathology , Endoplasmic Reticulum/physiology , Molecular Chaperones/metabolism , Proteasome Endopeptidase Complex/physiology , Protein Processing, Post-Translational/physiology , Analysis of Variance , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Cholera Toxin/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dystonia Musculorum Deformans/genetics , Fibroblasts , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Inbred C57BLABSTRACT
A case of 34 weeks pregnancy along with a large abdominal mass presented in emergency with a constant dull abdominal pain and premature labour. After delivery of the baby the dull ache persisted, prompting a laparotomy in the first postnatal week. A large cyst, arising from the left renal area and occupying almost the whole left side of the abdomen, was removed. Histopathology showed it to be an endometriotic cyst. The patient made an uneventful recovery.
