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First published online 3 August 2004
doi: 10.1242/jcs.01276

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Sialic acid residues on astrocytes regulate neuritogenesis by controlling the assembly of laminin matrices

¹ Departamento de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21941-590, Brazil
² Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21941-590, Brazil
³ Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21941-590, Brazil
⁴ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21941-590, Brazil

View larger version (66K): [in a new window]   Fig. 1. Astrocytes derived from embryonic or newborn animals produce morphologically different laminin matrices. Confluent monolayers of astrocytes isolated at embryonic day 16 (A,C-E) or at birth (B,F,G) were quickly fixed to preserve the associated extracellular matrix and processed for laminin immunocytochemistry (white in panels A and B and red in panels C-G). Cell nuclei were labeled with DAPI (blue staining). Note that laminin on E16 monolayers presented a periodical shape, made of polygons, which are better visualized in panels A and E (amplification of the field delimited by the larger frame in panel C). The P0 matrix forms webs that protrude from the cell surface (best seen in B and F). Panel D (field delimited by the smaller frame in C) shows the delicacy of the laminin mesh secreted by embryonic astrocytes, not comparable to the dense protein aggregates observed on P0 (G; amplification of the field delimited by the frame in F). Bars, 50 µm.

View larger version (66K): [in a new window]   Fig. 2. Neurite outgrowth is more pronounced on monolayers of embryonic than on monolayers of newborn astrocytes. Embryonic neurons were plated over confluent monolayers of previously established astrocyte cultures derived from E16 (A) or P0 (B) cortices. In A neurites are long and cell bodies are separated from each other. In B neurites are shorter and cell bodies tend to form clusters (arrows). Panels C and D show quantitative analyses of data obtained in panels A (black bars) and (B) (white bars). Quantification was performed for 100 neurons, randomly chosen. *P<0.001. Bar, 50 µm.

View larger version (25K): [in a new window]   Fig. 3. Astrocytes derived from E16 or P0 present different surface potentials. After mechanical dissociation of monolayers, astrocytes derived from E16 () or P0 () had their zeta () measured by cell electrophoresis. Values of Pzeta at different pHs are plotted in the main panel. The inset shows values of measured immediately after treatment with neuraminidase and 30 or 60 minutes after removal of the enzyme. *P<0.01; **P<0.001.

View larger version (95K): [in a new window]   Fig. 4. Treatment of embryonic astrocytes with neuraminidase led to the release of laminin matrices. When E16 monolayers were fixed immediately after 30 minutes incubation with neuraminidase, no reactivity for laminin was detected (A). At this condition, cells remained attached to the substrate, as shown by DAPI staining (B). When cells were allowed to recover for 1 hour in culture medium after neuraminidase removal (C,D) a laminin matrix with the features of the P0 matrix was assembled by the monolayer (compare with panel B in Fig. 1). Bar, 50 µm.

View larger version (48K): [in a new window]   Fig. 5. Neurite outgrowth is reduced on neuraminidase treated embryonic astrocytes. Neurons were seeded onto confluent E16 astrocyte monolayers either non-treated (A) or digested with neuraminidase and allowed to recover for 1 hour (B). Arrows in panel B point to cell clusters similar to those formed by neurons plated on P0 monolayers (see Fig. 2). Panels C and D show quantitative analyses of data obtained in panels A (black bars) and B (white bars). Quantification was performed for 100 neurons randomly chosen. *P<0.001. Bar, 50 µm.

View larger version (81K): [in a new window]   Fig. 6. Laminin matrices assembled on artificial lipid films present distinguished morphologies. Laminin matrices were assembled directly onto glass coverslips at the conditions indicated in the figure (A,D,G,H) or onto lipid films previously adsorbed to the coverslips (B,C,E,F). After 12 hours, artificial matrices were fixed and processed for immunostaining with anti-laminin antibodies. Bulky protein aggregates are observed in panels A to C, while lace-like polymers are seen in panels E to G. Panels D and H show conditions at which aggregation does not occur and laminin adsorbs evenly on the coverslips, probably as individual trimers. Bar, 50 µm.

View larger version (18K): [in a new window]   Fig. 7. Neuritogenesis is more pronounced on artificial laminin matrices assembled over lipid films containing negative charges. Artificial laminin matrices were assembled on coverslips coated with increasing ratios of GM1 (A), GT1b (B) or the pool, Gmix (C) relative to the neutral phospholipid, phosphatidylcholine. Neurons were seeded on each matrix and the induced neuritogenesis was evaluated. Quantification was performed for 100 neurons randomly chosen. Results are expressed using the percentyl plot. *P<0.001.

View larger version (79K): [in a new window]   Fig. 8. Methyl-ß-cyclodextrin promotes release of the laminin matrix from the cellular surface. Astrocyte monolayers derived from E16 rats were labeled for laminin and DAPI before (A) or after treatment with Triton X-100 (B) or CD (C). Triton X-100, a detergent capable of dissolving phospholipids, permeabilized the cells, allowing for visualization of intracellular stocks of the protein. Conversely, CD, which affects membrane structures stabilized by cholesterol, caused complete release of laminin from the monolayers. Insets show DAPI labeling in the respective fields. Bar, 50 µm.

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