José Garcia-Abreu

Amyloid-β binds to the extracellular cysteine-rich domain of frizzled and inhibits wnt/β-catenin signaling

The amyloid-β peptide (Aβ) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Aβ-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Aβ binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Aβ and are homologous to neuronal receptors putatively involved in Aβ interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Aβ with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Aβ and Fz. The results show that Aβ binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Aβ binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Aβ-binding site in Fz is homologous to cSP5 and that this is a relevant target for Aβ-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Aβ with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

Targeting the neurotoxic species in Alzheimer’s disease: inhibitors of Aβ oligomerization

In the past two decades, a large body of evidence has established a causative role for the β‐amy‐loid peptide (Aβ) in Alzheimers disease (AD). However, recent debate has focused on whether amyloid fibrils or soluble oligomers of Aβ are the main neurotoxic species that contribute to neurodegeneration and dementia. Considerable early evidence has indicated that amyloid fibrils are toxic, but some recent studies support the notion that Aβ oligomers are the primary neurotoxins. While this crucial aspect of AD pathogenesis remains controversial, effective therapeutic strategies should ideally target both oligomeric and fibrillar species of Aβ. Here, we describe the anti‐amyloido‐genic and neuroprotective actions of some di‐ and tri‐substituted aromatic compounds. Inhibition of the formation of soluble Aβ oligomers was monitored using a specific antibody‐based assay that discriminates between Aβ oligomers and monomers. Thioflavin T and electron microscopy were used to screen for inhibitors of fibril formation. Taken together, these results led to the identification of compounds that more effectively block Aβ oligomerization than fibrillization. It is significant that such compounds completely blocked the neurotoxicity of Aβ to rat hippocampal neurons in culture. These findings provide a basis for the development of novel small molecule Aβ inhibitors with potential applications in AD.—De Felice, F. G., Vieira, M. N. N., Saraiva, L. M., Figueroa‐Villar, J. D., Garcia‐Abreu, J., Liu, R., Chang, L., Klein, W. L., Ferreira, S. T. Targeting the neurotoxic species in Alzheimers disease: inhibitors of Aβ oligomerization.

Inhibition of Alzheimer’s disease β-amyloid aggregation, neurotoxicity, and in vivo deposition by nitrophenols: implications for Alzheimer’s therapy

Alzheimers disease (AD) is a major public health problem, and there is currently no clinically accepted treatment to cure it or to stop its progression. Fibrillar aggregates of the β–amyloid peptide (Aβ) are major constituents of the senile plaques found in the brains of AD patients and have been related to AD neurotoxicity. Here it is shown that nitrophenols prevent aggregation and cause disaggregation of Aβ fibrils and that they strongly prevent the neurotoxicity of Aβ to rat hippocampal neurons in culture. Furthermore, by using an in vivo model system of cerebral amyloid deposition, it is shown that nitrophenols cause a marked reduction in the volume occupied by amyloid deposits in the hippocampi of rats. These results raise the possibility that nitrophenols or their derivatives may be useful lead compounds for the development of drugs to prevent the neurotoxicity and deposition of Aβ in AD.

Neurons induce GFAP gene promoter of cultured astrocytes from transgenic mice

In order to investigate the influence of neuron‐glia interaction on astrocyte differentiation, we used a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP linked to the β‐galactosidase (β‐gal) reporter gene. Addition of embryonic cerebral hemisphere (CH) neurons to transgenic CH astrocyte monolayers increased by 50–60% β‐gal positive cell number. Such event was dependent on the brain regional origin of the neurons and was followed by an arrest of astrocytes from the cell cycle and induction of glial differentiation. Time‐course assays demonstrated that maximum effect was observed after 24 h of coculture. Addition of conditioned medium (CM) derived from CH neurons also increased β‐gal positive CH astrocytic cell number. However, such CM had no effect on midbrain and cerebellum astroglia. Together, these data suggest that neurons secrete brain region‐specific soluble factors which induce GFAP gene promoter, as measured by β‐gal expression, thus suggesting that neuron‐glia interaction might induce the astrocytic differentiation program. GLIA 26:97–108, 1999.

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.

Glial cells with differential neurite growth-modulating properties probed by atomic force microscopy.

Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. Thus, an important choice point for developing axons is the Central Nervous System (CNS) midline. Crossing of this choice point is influenced by highly conserved, soluble or membrane-bound molecules such as the L1 subfamily, laminin, netrins, slits, semaphorins, Eph-receptors and ephrins, etc. Furthermore, there is much circumstantial evidence for a role of proteoglycans (PGs) or their glycosaminoglycan (GAG) moieties on axonal growth and guidance, most of which was derived from simplified models. A model of intermediate complexity is that of cocultures of young neurons and astroglial carpets (confluent cultures) obtained from medial and lateral sectors of the embryonic rodent midbrain soon after formation of its commissures. Neurite production in these cocultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exerted an inhibitory or non-permissive effect on neuritic growth that was correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment with GAG lyases shows minor effects of CS and discloses a major inhibitory or non-permissive role for HS. The results are discussed in terms of available knowledge on the binding of HSPGs to interative proteins and underscore the importance of understanding glial polysaccharide arrays in addition to its protein complement for a better understanding of neuron-glial interactions.

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.