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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02716

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Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation

¹ Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan
² Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan
³ Department of Neurosurgery, Graduate School of Medicine, Jikei University School of Medicine, Minato-ku, Tokyo, 105-8461, Japan
⁴ Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392, Japan

View larger version (53K): [in a new window]   Fig. 1. Antibody specificity and expression of UCH-L1 in the ventricular zone at E13. UCH-L1 expression was detected using immunohistochemistry with anti-PGP9.5. UCH-L1 is not detected in the brain of gad mice at E13 (A,B) but is strongly expressed in heterozygous littermates (A,B). Confocal microscopic images of coronal sections of gad mice and heterozygous littermates were double stained with antibodies for the progenitor marker nestin and UCH-L1 (PGP9.5) (A) or for the early neuronal marker tubulin ß III (TuJ1) and UCH-L1 (B). Long radial fibers are indicated by arrowheads, and various phases of progenitor cells are indicated by arrows (A). TuJ1-positive, migrating neuronal cells are indicated by arrowheads (B). MZ, marginal zone; VZ, ventricular zone. Bars, 40 µm.

View larger version (113K): [in a new window]   Fig. 2. Change in UCH-L1 expression pattern in the developing mouse brain. Cryosections of the brain at E14 and E16 were double stained with UCH-L1 and the neural progenitor marker nestin (A) or early neuronal marker TuJ1 (B). Unlike with UCH-L1, staining patterns for TuJ1 and nestin do not change between E14 and E16. At E14, UCH-L1 expression is higher in the VZ than in the MZ. At E16, higher expression of UCH-L1 is reciprocally detected in the CP. By contrast, at both E14 and E16, nestin is highly expressed in the VZ, and TuJ1 expression is higher in the MZ/CP. Fluorescence intensities per field (1700 µm2) were measured in each layer of the E14 and E16 brain and are shown to the right. Bars, 80 µm. (C,D) Higher-magnification images from A,B of UCH-L1 expression in the E14 and E16 brain: UCH-L1 and nestin (C); UCH-L1 and TuJ1 (D). UCH-L1 and nestin are co-expressed in the VZ at E14 and E16. Nestin is expressed only in radial glial fibers (arrowheads) of the CP but not in neurons. UCH-L1 expression level is high. A representative cell with a high level of UCH-LI expression is indicated by a white arrow and one with low expression is indicated by a yellow arrow (C). An early neuronal marker, TuJ1, was expressed in both migrating (arrowheads) and mature neurons (D). CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone. Bars, 40 µm.

View larger version (59K): [in a new window]   Fig. 3. Nestin and UCH-L1 expression in undifferentiated and differentiating NPCs at 2, 6, 12, 24 and 48 hours. (A) NPCs were immunolabeled with antibodies against nestin and UCH-L1 in the proliferating phase (+bFGF; at 48 hours) or the differentiation phase (-bFGF; 2, 6, 24, 48 hours). Cultures were counterlabeled with Hoechst nuclear dye to facilitate cell quantification. (B) Quantitative analysis of the percentage of cells stained with each antibody. Nestin-positive cells gradually decrease as differentiation proceeds. The UCH-L1 expression level is both high (arrowheads) and low (arrows) in nestin-positive cells at 6 hours. Each experiment was analyzed by counting cells in three independent wells at the indicated times. The experiments were repeated at least two times. Bars, 50 µm.

View larger version (69K): [in a new window]   Fig. 4. UCH-L1 expression in neurogenesis. NPCs were immunolabeled with antibodies against TuJ1 and UCH-L1. Cultures were counterlabeled with Hoechst nuclear dye to facilitate cell quantification. Quantitative analysis of the percentage of cells stained with each antibody. (A) In the proliferating phase (+bFGF; at 48 hours) or the differentiation phase (-bFGF; 2, 12, 24, 48 hours), most TuJ1-positive cells co-express UCH-L1. The UCH-L1 expression level is both high and low in TuJ1-positive cells at 48 hours. (B) Quantitative analysis of the percentage of cells stained with each antibody. The number of TuJ1-positive cells gradually increased in the differentiating phase (-bFGF; B). Each experiment was analyzed by counting cells in three independent wells at the indicated times. The experiments were repeated at least two times. Bars, 50 µm.

View larger version (34K): [in a new window]   Fig. 5. Morphological identification of subpopulations of cultured NPCs at 6 and 12 hours after induction of differentiation. Differentiating NPCs were double stained with UCH-L1 and nestin. For the quantification depicted in A, differentiating NPCs stained with UCH-L1 and nestin were classified as long, short or round (see text). For the quantification depicted in B, differentiating NPCs were classified based on three kinds of cell morphology: unipolar, bipolar, or multipolar.

View larger version (28K): [in a new window]   Fig. 6. The induction of short processes depends on the interaction between UCH-L1 and monoubiquitin. (A) FLAG-tagged WT UCH-L1, C90S UCH-L1, D30A UCH-L1 and GFP (all in the pCI-neo vector) were transfected into NPCs. Antibodies against the FLAG-tag were used to detect transfected UCH-L1. The green staining shows transfected cells and the red staining shows endogenous nestin. Transient transfection of each construct was performed under proliferating conditions. At 48 hours after transfection, bFGF was removed for 12 hours before the cultures were immunostained. The lengths of nestin-positive processes in immunostained cells were measured. Asterisks indicate differences from the value of GFP-transfected NPCs at *P<0.05 and ***P<0.001. Bars, 80 µm. (B) Visualization of recombinant 6HN-tagged UCH-L1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining (B, left). UCH-L1 hydrolase activity was measured by Ub-AMC hydrolysis. Enzyme concentration was 4.3 nM, and substrate concentration was 700 nM. Initial velocity data was used to determine the values for relative hydrolase activity of UCH-L1 (B, right). (C) UCH-L1 co-immunoprecipitated with Ub. Cytosolic extracts from NIH-3T3 cell lines stably expressing HA-tagged WT UCH-L1 and mutants thereof were immunoprecipitated using anti-HA and immunoblotted with anti-HA antibody or anti-Ub antibody.

View larger version (81K): [in a new window]   Fig. 7. A comparative experiment of gad mice and heterozygous littermates. The experiment compared gad mice (A,B) with a transfection study using FLAG-tagged WT UCH-L1, C90S UCH-L1, D30A UCH-L1 and GFP (mock) into gad-mouse-derived NPCs (C). The lengths of nestin-positive processes in immunostained cells were measured. NPCs from gad mice had longer nestin-positive processes compared with the control (A,B). (C) At 48 hours after transfection, bFGF was removed for 12 hours before the cultures were immunostained. The lengths of nestin-positive processes in immunostained cells were measured. Asterisks indicate differences from the value of GFP-transfected NPCs at **P<0.01 and ****P<0.0001. Bar, 50 µm.

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