title: Isolation and characterization of neural stem cells from dystrophic mdx mouse.
abstract: The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Annese
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Corsi
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Ruggieri
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Tamma
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
authors.forename: Christian
authors.initials: C
authors.lastname: Marinaccio
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Picocci
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
authors.forename: Mariella
authors.initials: M
authors.lastname: Errede
authors.affiliation: Department of Emergency and Transplantation, Section of Hematology, University of Bari Medical School, Bari, Italy.
authors.forename: Giorgina
authors.initials: G
authors.lastname: Specchia
authors.affiliation: Department of Bioscience, Biotechnology and Pharmacological Sciences, Section of Pharmacology, University of Bari, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: De Luca
authors.affiliation: Department of Internal Medicine and Oncology, University of Bari Medical School, Bari, Italy.
authors.forename: Maria Antonia
authors.initials: MA
authors.lastname: Frassanito
authors.affiliation: Department of Internal Medicine and Oncology, University of Bari Medical School, Bari, Italy.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Desantis
authors.affiliation: Department of Internal Medicine and Oncology, University of Bari Medical School, Bari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Vacca
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy; National Cancer Institute "Giovanni Paolo II", Bari, Italy. Electronic address: domenico.ribatti@uniba.it.
authors.forename: Domenico
authors.initials: D
authors.lastname: Ribatti
authors.affiliation: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy. Electronic address: beatrice.nico@uniba.it.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Nico
mesh_terms: D000071916:AC133 Antigen / Q000378:metabolism; D053687:Adult Stem Cells / Q000166:cytology / Q000378:metabolism; D000818:Animals; D051401:Aquaporin 4 / Q000378:metabolism; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D049030:Dystroglycans / Q000378:metabolism; D016189:Dystrophin / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008565:Membrane Proteins; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D009124:Muscle Proteins; D009137:Muscular Dystrophy, Animal / Q000235:genetics / Q000473:pathology*; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000166:cytology / Q000648:ultrastructure; D025801:Ubiquitin / Q000378:metabolism; D000098854:Kcnj10 Channel
chemical_list: D000071916:AC133 Antigen; D051401:Aquaporin 4; D002135:Calcium-Binding Proteins; D016189:Dystrophin; D005904:Glial Fibrillary Acidic Protein; D000098854:Kcnj10 Channel; D008565:Membrane Proteins; D009124:Muscle Proteins; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D025801:Ubiquitin; C085339:apo-dystrophin 1; C091825:syntrophin alpha1; D049030:Dystroglycans; D046988:Proteasome Endopeptidase Complex
keywords: Blood-brain barrier; Dystrophic mice; Dystrophin-associated proteins; Neural stem cells; Neurospheres