Glaucia R. Onofre

Contribution of heparan sulfate to the non-permissive role of the midline glia to the growth of midbrain neurites

Radial glial cells and astrocytes are heterogeneous with respect to morphology, cytoskeletal‐ and membrane‐associated molecules and intercellular interactions. Astrocytes derived from lateral (L) and medial (M) midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in coculture (Garcia‐Abreu et al. J Neurosci Res 40:471, 1995). There is a correlation between these abilities and the differential patterns of laminin (LN) organization that is fibrillar in growth‐permissive L astrocytes and punctate in the non‐permissive M astroglia (Garcia‐Abreu et al. NeuroReport 6:761, 1995). There are also differences in the production of glycosaminoglycans (GAGs) by L and M midbrain astrocytes (Garcia‐Abreu et al. Glia 17:339, 1996). We show that the relative amounts of the glycoproteins laminin LN, fibronectin (FN) and tenascin (TN) are virtually identical in L and M glia, thus, confirming that an abundant content of LN is not sufficient to promote neurite growth. To further analyze the role of GAGs in the properties of M and L glia, we employed enzymatic degradation of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS). Treatment with chondroitinase has little effect on the non‐permissive properties of M glia but reduces the growth‐supporting ability of L glia. By contrast, heparitinase I produces no significant changes on L glia but leads to neurite growth promotion by M glia. Taken together, these results suggest that glial CS helps to promote neurite growth and, more importantly, they indicate that a HS proteoglycan is, at least, partially responsible for the non‐permissive role of the midline glia to the growth of midbrain neurites. GLIA 29:260–272, 2000.

Compartmental distribution of sulfated glycosaminoglycans in lateral and medial midbrain astroglial cultures

Sulfated glycosaminoglycans (S‐GAGs) were isolated from the pericellular (P), intracellular (I), and extracellular (E) compartments of astrocytes cultures from lateral (L) and medial (M) sectors of embryonic mouse midbrain; these sectors differ in their ability to support neurite growth (L, permissive, M, non‐permissive for growth) and laminin deposition patterns (L, fibrillar; M, punctate pattern). The total amount of S‐GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment of M glia. Both glial cultures showed heparan sulfate (HS) in the three cellular compartments but chondroitin sulfate (CS) GAGs were vestigial in I and P compartments of L glia. Our results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments. Together with other data (Garcia‐Abreu et al: J Neurosci Res 40:471, 1995; Garcia‐Abreu et al: Neuroreport 6:761, 1995), the present results suggest that this heterogeneous features might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons and may also be involved in complex ways in the guidance of axons at the brain midline.

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of beta-D-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose-effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.

Heparan sulfate glycosaminoglycan expression in the intestinal tract and ovary of fully engorged adult females of the cattle tick Boophilus microplus and in their laid eggs.

Departamento de Bioqu´imica Medica, Instituto de Ciˆencias Biomedicas, Centro de Ciˆencias da Saude,Universidade Federal do Rio de Janeiro, 21941-590, Caixa Postal 68041, Rio de Janeiro, BrazilReceived 22 April 2003; received in revised form 30 May 2003; accepted 18 June 2003Keywords: Sulfated glycosaminoglycans; Heparan sulfate; Chondroitin sulfate; Tick; Boophilus microplus

Astroglial cells derived from lateral and medial midbrain sectors differ in their synthesis and secretion of sulfated glycosaminoglycans

Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and beta-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted approximately 2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of beta-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth.

Composition of urinary glycosaminoglycans in a patient with relapsing polychondritis.

OBJECTIVES Several investigators have reported an increase in urinary glycosaminoglycans (GAGs) in patients with relapsing polychondritis (RP). The aim of this investigation is to analyze the composition and structure of urinary GAGs from a Brazilian patient with RP. DESIGN AND METHODS The identification and structural analyses of the GAGs were made by electrophoresis and degradation with specific enzymes and identification of their disaccharides products by HPLC chromatography. RESULTS The disaccharide products formed from RP urinary chondroitin sulfate (CS) by action of chondroitin ABC lyase showed a substantial relative increase of nonsulfated disaccharides with a relative decrease of 6-sulfated disaccharides compared to control subjects. In addition, a significant change of the ratio of CS and heparan sulfate was also observed in the RP patient. CONCLUSION The RP patient analyzed has shown a structural anomaly of the urinary CS and this may contribute to the diagnosis of this disease.

Sulfated proteoglycans as modulators of neuronal migration and axonal decussation in the developing midbrain

Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.