Toxin-Based Models to Investigate Demyelination and Remyelination.

Clinical myelin diseases, and our best experimental approximations, are complex entities in which demyelination and remyelination proceed unpredictably and concurrently. These features can make it difficult to identify mechanistic details. Toxin-based models offer lesions with predictable spatiotemporal patterns and relatively discrete phases of damage and repair: a simpler system to study the relevant biology and how this can be manipulated. Here, we discuss the most widely used toxin-based models, with a focus on lysolecithin, ethidium bromide, and cuprizone. This includes an overview of their respective mechanisms, strengths, and limitations and step-by-step protocols for their use

Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors

Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS)--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS

AC3 detection in primary cilia of human developing organs.

<p>AC3 detection in primary cilia of human developing organs.</p

AC3 expression in adult mouse cilia.

<p>(A) Brain: note the long AC3 positive cilia in grey matter and the absence of AC3 positive cilia in white matter (corpus callosum). (B) Peripheral layers of the olfactory bulb: note the short AC3-positive cilia in the olfactory nerve layer (arrow) as compared to the long cilia of cells around the olfactory glomeruli (arrow head). (C) Seminiferous tubule: AC3 is present in the developing acrosome of round spermatids. Abbreviations: GM, grey matter; WM, white matter; ONL, olfactory nerve layer; G, glomerulus; GL, glomerular layer; PL, plexiform layer.</p

AC3-positive cilia in human epithelia.

<p>(A) Olfactory epithelium in GW8 embryo; AC3-positive cilia on olfactory neurons (arrows). (B-C) Inner ear of a GW8 embryo. (B) Section through the endolymphatic duct (arrow) and semicircular ducts (arrow head). (C) Adjacent section showing AC3-positive primary cilia of apical epithelial cells lining the endolymphatic duct. (D-F) Kidney of a GW12 fetus. (D) Primary cilia are present on epithelial cells of the glomerulus and the distal tubule as well as on mesenchymal cells of the floculus and in interstitial cells. (E-F) AC3 is absent from apical epithelial cilia but present in mesenchymal cells cilia of the glomerular floculus and in the interstitium (F is a high magnification of E). (G-I) Pancreas of a GW12 fetus. (G) Hematoxylin-eosin stained section; only ducts but not secretory acini are present at this developmental stage. (H) Apical cilia of pancreatic duct are very long as compared to interstitial cells cilia. (I) Only cilia of cells around the ducts are AC3-positive (arrows). (J-L) liver of a GW12 fetus. (J) Portal space surrounded by the ductal plate (arrow) and small biliary ducts (arrow head). (K) Ductal plate cells (arrow) and the small biliary ducts cells (arrow head) have long primary cilia. (L) Absence of AC3 in epithelial cells cilium. (M-O) Stratified epithelia (M) Respiratory epithelium AC3 positive cilia of basal cells (arrows). (N-O) Oral epithelium. (N) Cilia in basal cells (arrow); presence of small nerve fibers in the lamina propria (arrow heads). (O) The cilia of basal cells are AC3 positive. Abbreviations: AC3: adenylate cyclase type III; Ac Tub: acetylated-tubulin; HE: Hematoxylin-eosin.</p

AC3-positive cilia in human and mouse tissues of mesenchymal origin during development.

<p>(A-D) Tissue sections from a GW12 human fetus. (A) Meckel’s cartilage: short AC3-positive cilia in perichondral cells and chondroblasts. (B) Bone trabecula: AC3-positive cilia are present in osteoblasts (arrow) and osteocytes (arrow head). (C) Myotubes. (D) Mesenchymal cells. (E, F) Adjacent sections of a mouse tooth at the bell stage. (E) Hematoxylin-eosin staining. (F) AC3 immunohistochemistry shows positive cilia on odontoblasts (o) and peripheral cells of the enamel organ (eo) but not in ameloblasts (a). Abbreviations: AC3: adenylate cyclase type III; HE: Hematoxylin-eosin.</p