David R. Canning

Chondroitin Sulfate Proteoglycans Are Associated with the Lesions of Alzheimer's Disease

Chondroitin sulfate proteoglycans (CSPG) are extracellular matrix proteins inhibitory to neurite outgrowth in vitro and correlated with decreased neurite outgrowth after CNS injury. Previously, heparan sulfate proteoglycan and dermatan sulfate proteoglycan have been shown to be associated with senile plaques (SPs) and neurofibrillary tangles (NFTs) but CSPG was not. In an immunocytochemical study, three monoclonal antibodies to different sulfation states of the chondroitin glycosaminoglycan were used to localize CSPG in cases of Alzheimers disease. Chondroitin 4-sulfate was found in both SPs and NFTs. An antibody to unsulfated chondroitin strongly immunostained intracellular NFTs and the dystrophic neurites of SPs. Chondroitin 6-sulfate was found in NFTs and the area around SPs. These results suggest that CSPG, in addition or as an alternative to beta-amyloid protein, could be responsible for the regression of neurites around senile plaques in Alzheimers disease.

β-Amyloid of Alzheimer's Disease Induces Reactive Gliosis That Inhibits Axonal Outgrowth

Pathological lesions in the brains of patients with Alzheimers disease (AD) are characterized by dense deposits of the protein beta-amyloid. The link between the deposition of beta-amyloid in senile plaques and AD-associated pathology is, at present, controversial since there have been conflicting reports on whether the 39-43 amino acid beta-amyloid sequence is toxic or trophic to neurons. In this report, we show that beta-amyloid peptide when presented as an insoluble substrate which mimics its conformation in vivo can induce cortical glial cells in vitro and in vivo to locally deposit chondroitin sulfate containing proteoglycan. In vitro the proteoglycan-containing matrix deposited by glia on beta-amyloid blocks the usual ability of the peptide to allow cortical neurons to adhere and grow. Chondroitin sulfate-containing proteoglycan was also found in senile plaques of human AD tissue. We suggest that an additional effect of beta-amyloid in the brain, which compounds the direct effects of beta-amyloid on neurons, is mediated by the stimulation of astroglia to become reactive. Once in the reactive state, glial cells deposit large amounts of growth-inhibitory molecules within the neuropil which could impair neuronal process survival and regeneration leading to neurite retraction and/or dystrophy around senile plaques in AD.

A potent inhibitor of neurite outgrowth that predominates in the extracellular matrix of reactive astrocytes.

In a model of astrogliosis in vitro, cultured cortical astrocytes were triggered into a functionally reactive state by an immobilized fragment of the β‐amyloid peptide. Induced astrocytes produced an extracellular matrix that inhibited the outgrowth of embryonic CNS axons. Within the extracellular matrix deposited by reactive astrocytes, we found an overall increase in the deposition of chondroitin sulphate that accounted for the inhibition. Specifically, we have detected an increased biosynthesis of a small chondroitin/heparan sulphate proteoglycan that is a potent inhibitor of axon outgrowth. We further suggest that this proteoglycan, or related molecules yet to be discovered, may play a role in gliosis‐mediated regenerative failure of CNS axons.

Regional Differences in Reactive Gliosis Induced by Substrate-Bound β-Amyloid

Abstract Regions of gliosis surround deposits of (β-amyloid peptide (βAP) in senile plaques of Alzheimers disease (AD). The association between reactive astrocytes and βAP in senile plaques is most pronounced in cortex and hippocampus but not at other anatomical sites of βAP deposition. We hypothesized that this region-specific pathology in AD could be attributed to differences in glial reactivity in different parts of the central nervous system (CNS). To test this hypothesis, we assayed astrocytes from cerebral cortex, hippocampus, cerebellum, and spinal cord for cellular responsiveness to substrate-bound βAP in vitro . Astrocyte reactivity was monitored by morphological changes, increased deposition of chondroitin sulfate proteoglycan-containing matrix, and alterations in proteoglycan metabolism. Based on these criteria, only cortical and hippocampal astrocytes showed marked reactivity to immobilized βAP. In cortical and hippocampal cultures only, immobilized βAP resulted in increased total radiosulfate incorporation into proteoglycans which was mainly found in the cell/matrix rather than in the media-associated compartment. There were also differences in the proteoglycan synthesis patterns of astrocyte cultures isolated from these CNS regions. These findings suggest that (1) astrocytes are regionally heterogenous in their reactive response to βAP and (2) that specific molecules, in addition to βAP, may exist following trauma or disease which trigger reactive states in astroglia in the cerebellum or spinal cord. These local differences in the interaction between βAP and surrounding astrocytes may play a role in the region-specific pathogenesis of Alzheimers disease.

TEACHING THE STRUCTURAL NATURE OF BIOLOGICAL MOLECULES: MOLECULAR VISUALIZATION IN THE CLASSROOM AND IN THE HANDS OF STUDENTS

The use of molecular visualization software has made a tremendous impact in the biochemistry and cell biology classroom. Instructors no longer have to rely on static images in textbooks to teach the structural nature of biological molecules. The emergence of many different molecular graphics programs and technology-based classrooms has enhanced the ability of instructors to teach structural concepts such as noncovalent interactions and levels of organization in proteins. Many web-based tutorials are also available for instructors to use during lecture or for students to explore outside of the classroom. Students can also obtain hands-on experience with the graphics programs to explore the structural aspects of macromolecular systems. This report shows that students involved in visualization projects become skilled at identifying various structural motifs they have discussed in class or are discovering for the first time. This student-centered approach enhances the ability of students to comprehend structural concepts and to realize the importance of weak interactions in the structure of large molecules. [Chem. Educ. Res. Pract. Eur.: 2001, 2, 109-122]

Regulation of epiblast cell movements by chondroitin sulfate during gastrulation in the chick

Avian gastrulation is dependent on the ingression of outer layer cells into the interior of the embryo by means of a transient structure referred to as the primitive streak. As the growing streak progresses through the central area pellucida of the blastoderm, selective de‐epithelialization of epiblast cells results in the initial migratory cells of the primitive mesoderm and endoderm. Here, we have examined the possibility that extracellular matrix molecules of the epiblast basal lamina influence the selection of streak‐specific epiblast cells. By using whole embryo culture, we have found that removal of chondroitin sulfate glycosaminoglycans at gastrulation stages leads to defective streak formation. In situ hybridization with streak‐specific markers in these embryos reveals ectopic patterns of gene expression, suggesting that differentiation of primitive streak precursors in the pregastrula epiblast is independent of normal streak morphogenesis. In addition, in vitro assays with chondroitin sulfate containing matrices suggest that specific cells of the epiblast are inhibited from joining the streak during gastrulation. Taken together, these results indicate that the presence of chondroitin sulfate in the epiblast basal lamina facilitates the allocation of cells to the primary germ layers by preventing ectopic axis formation.

Chondroitin sulfate effects on neural stem cell differentiation.

We have investigated the role chondroitin sulfate has on cell interactions during neural plate formation in the early chick embryo. Using tissue culture isolates from the prospective neural plate, we have measured neural gene expression profiles associated with neural stem cell differentiation. Removal of chondroitin sulfate from stage 4 neural plate tissue leads to altered associations of N-cadherin-positive neural progenitors and causes changes in the normal sequence of neural marker gene expression. Absence of chondroitin sulfate in the neural plate leads to reduced Sox2 expression and is accompanied by an increase in the expression of anterior markers of neural regionalization. Results obtained in this study suggest that the presence of chondroitin sulfate in the anterior chick embryo is instrumental in maintaining cells in the neural precursor state.

Cell adhesion properties of neural stem cells in the chick embryo.

The nervous system of vertebrates is derived from an early embryonic region referred to as the neural plate. In the chick embryo, the neural plate is populated by neural stem cells specified from the epiblast shortly after the onset of gastrulation. Accompanying the formation of the plate, chondroitin sulfate glycosaminoglycans are expressed in the basal extracellular matrix. We describe in vitro experiments measuring cell adhesion of epiblast cells during the formation of the neural plate. Our findings may suggest that neural stem cells are set apart from non-neural epiblast by changes in relative cell-cell and cell-substrate adhesion. Specifically, changes in cell adhesion separating neural stem cells from the non-neural epiblast may be augmented by the presence of exogenous chondroitin-6-sulfate in the epiblast basal lamina at the time neural progenitors are specified in the epiblast.

A Comparison Between Antigen-Laden Cells and Dendritic Cells in Afferent and Efferent Lymph

The importance of antigen presenting cells in the initiation of an immune response has focused attention on two contrasting cell types — the antigen-laden (Ag-L) cell (a macrophage-1ike cell) and the dendritic cell (DC) alias the “veiled” cell or interdigitating cell of the lymph node (LN) paracortex. Whereas the DC is recognized by a high surface density of Ia molecules and can be found in afferent lymph,1–4 the Ag-L cell is found in low frequency (<0.01%) in the efferent lymph of the thoracic duct, lacks Ia6 and appears to transport injected antigens.7 Functionally, the DC stimulates T cell proliferation3,8,9 while the Ag-L cell is active in triggering specific memory cells into antibody synthesis on adoptive transfer. 5,6