Modifications of perineuronal nets to regulate plasticity Heleen Merel van 't Spijker Perineuronal nets (PNNs) are macromolecular structures formed by neurons after closure of critical periods of plasticity. During development, the central nervous system (CNS) goes through critical periods of plasticity; a period when substantial changes occur to adapt to the environment, during which many synapses are formed and also discarded. When a region of the CNS has finished its development and reached an efficient neuronal circuit, the capacity for plasticity needs to be reduced to preserve the formed circuit. PNNs are formed around neurons during this period of reduced plasticity. PNNs consist of a backbone of hyaluronan, bound by chondroitin sulfate proteoglycans (CSPGs). Here, I present my studies on the possible modifications of PNNs to regulate plasticity. Firstly, I have investigated the potential use of 4-methylumbelliferone (4-MU) to reduce PNN formation in vivo. 4-MU reduces the formation of hyaluronan. Since hyaluronan is the backbone of PNNs, I hypothesized 4-MU treatment would reduce PNN formation. For this study, I developed a method to orally administer 4-MU to rats. Subsequently, I investigated whether 4-MU treatment can improve recovery of rats after spinal cord injury, both with behavioural tests and with immunohistochemistry. Secondly, I have investigated a new binding partner of PNNs, neuronal pentraxin 2 (Nptx2). Nptx2 is secreted by neurons and regulates AMPA receptor diffusion. Nptx2 knockout mice show a prolonged critical period of plasticity in the visual cortex. Here, I have identified Nptx2 as a new binding partner of PNNs. Nptx2 is found in isolated PNN protein preparations and is removed from the surface of neurons by digestion of PNNs with chondroitinase ABC. I also determined Nptx2 facilitates PNN formation in vitro. Addition of Nptx2 to the medium of cortical neurons leads to an increase of neurons that start to form PNNs, as well as an increase in size and density of PNNs. These findings indicate Nptx2 may be used as a modulator of PNNs. Thirdly, I investigated the interaction between Nptx2 and PNNs. I developed a sandwich ELISA to determine which glycan chains from PNNs bind to Nptx2. Nptx2 binds to chondroitin sulfate E and hyaluronan. To investigate the binding properties of Nptx2, I performed quartz crystal microbalance with dissipation monitoring for Nptx2 films. Furthermore, I developed crystals of purified Nptx2 and hyaluronan for x-ray crystallography. The here presented results provide new insights in potential approaches to modulate PNN formation. Both lines of research provide a further understanding of the factors which regulate PNNs and may allow for the development of treatments for PNN related disorders.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年4月5日 西田佳弘氏の博士論文として提出された

The extracellular environment of the central nervous system (CNS) through which neuritic processes must traverse during development or after injury is complex, and may vary from stabile conditions to a milieu favorable for neural plasticity and growth. The extracellular space in the CNS accounts for about 20% of brain volume and is composed of aggregating complexes of several different extracellular matrix (ECM) molecules. The ECM supports neural networks and acts as a barrier for neurite extention, depending on the type of molecules involved and the various signals they induce. One mechansim that may produce an environment favoring plasticity is the proteolytic cleavage of ECM. Brevican belongs to the lectican family of aggregating, chondroitin sulfate-containing proteoglycans (CSPGs) and is abundant in brain ECM complexes. It is localized peri-synaptically, inhibits neurite outgrowth, and is thought to stabilize synaptic networks in the adult. Interestingly, a significant proportion of brevican in the CNS is observed as a fragment of the protein core formed by proteolytic cleavage. Endogenous matrix-degrading proteinases, such as the MMPs (matrix metalloproteinases) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), cleave brevican and other lecticans potentially promoting neural plasticity. Cleavage of brevican and similar lectican family members may "loosen" the aggregated complexes and change the extracellular environment to one that is more permissive toward neural plasticity. After injury, during inflammation or with disease, alterations in the ECM may influence development and/or progression of neurological disease. The purpose of these studies was to investigate the catabolism of brevican in the ECM and its potential role in neural plasticity under each of these influences, taking an in depth look at how brevican is processed after (1) undergoing a classical model of neural plasticity, the entorhinal cortex lesion (ECL); (2) a disease state that is thought to have dysregulated neural and synaptic plasticity; and (3) how brevican catabolism and neural plasticity is effected by deleting the protease responsible for the cleavage of lecticans in a mouse model. Overall, these experiments provide evidence that the proteolytic cleavage of brevican, and lecticans in general, may play an important role in the regulation of neural plasticity.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.

Cultured bovine aortic endothelial (BAE) cells were found to synthesize and secrete heparan sulfate proteoglycans (HSPG), which bound basic fibrobalst growth factor (bFGF). bFGF is a known mitogen for vascular smooth muscle cells, and is indicated to have a role in some proliferative vascular disorders. In the present study, we have purified proteoglycans from BAE cells conditioned media (BAE PG), and further separated the PG into two fractions, PG-I and PG-II, by ion exchange chromatography on a Q-Sepharose column using a linear salt gradient (0.15 M to 1.2 M). PG-I and PG-II elute at 0.85M salt and 0.1M salt respectively. BAE PG is primarily composed of heparan sulfate, which is accessible to the digestion of Heparinase I/III and nitrous acid treatment; and a small amount of chondroitin sulfate, which can be digested by Chondroitinase ABC. Gel filtration chromatography (Sepharose CL-2B and CL-4B columns) showed that BAE PG consisted of two different sized peaks, and had an average molecular weight of approximately 5 x 10⁵ Da. SDS-PAGE with silver staining indicated that BAE PG had two core proteins with estimated sizes of 300kDa and 320kDa, which corresponded to the core protein of PG-I and PG-II respectively. Western blotting with anti-perlecan primary antibody recognized the core proteins of BAE PG. Size exclusion chromatography (Sepharose CL-6B column) following β-elimination showed that BAE PG had GAG chains with an estimated size less than 2 x 10⁵ Da. A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, K_D, of (1.6 ± 0.8) x 10⁻¹⁰ M; the dissociation rate constant, k_r, of 0.01 min⁻¹; the association rate constant, k_f, of 6.2 x 10⁷ M⁻¹min⁻¹ and the total binding sites of the proteoglycan, R_T, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds. / Master of Science

Current articular cartilage tissue engineering endeavors, using synthetic polymers as scaffolds, have been somewhat successful. However, the use of these materials has not yielded a satisfactory, functional replacement for articular cartilage. Therefore, this project focuses on an alternative to these materials, chitosan, which is a naturally occurring biopolymer. The first project objective was to fabricate and analyze bulk, porous chitosan scaffolds, based on total porosity, average pore diameter, mechanical integrity, and degradation susceptibility. Secondly, scaffolds were evaluated in terms of their ability to support neochondrogenesis, including assessments of cell attachment and viability, cell morphology, and the biosynthesis of proteoglycan and type-II collagen-rich extracellular matrix. Results indicated that chitosan scaffolds possessing an interconnecting, porous structure could be easily created through a simple freezing and lyophilization process, and these scaffolds did support neochondrogenesis. Results suggest chitosan may be a useful alternative to synthetic polymers for use in cartilage tissue engineering applications.

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Despite the effort to optimize soft contact lens performance, almost half of the 140 million contact lens wearers worldwide experience symptoms of ocular dryness and discomfort, especially towards the end of the day. These symptoms are attributed to reduced compatibility between the contact lens and the ocular surface and are the main reason for contact lens discontinuation. As the interactions of the contact lens-eye interface are dynamic, the surface properties play a key role in improving ocular compatibility, comfort and overall performance of contact lenses. One promising method to reduce adverse interfacial interactions between the contact lens and the ocular surface is to modify the contact lens surface with a biomimetic layer inspired by the ocular surface and the tear film. Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan naturally found in the ocular environment providing ocular hydration and lubrication. Proteoglycan 4 (PRG4), a mucin-like glycoprotein naturally produced at the ocular surface contributes to natural lubrication during blinking and to tear film stability. Surface modification with HA or PRG4 has been shown to result in improved wetting, lubricating and antifouling properties. Moreover, HA and PRG4 have been previously found to interact and synergistically reduce friction further. In the current work, novel HA and PRG4-grafted soft contact lens surfaces were prepared, and the impact of the surface tethered layer on important contact lens properties was assessed. Furthermore, the potential synergistic effect between HA and rhPRG4 on the examined properties was evaluated. Surface immobilization of HA on model conventional (pHEMA) and silicone (pHEMA-co-TRIS) hydrogel contact lenses was achieved by thiol-ene “click” chemistry, while full-length recombinant human PRG4 (rhPRG4) was surface grafted via carbonyldiimidazole (CDI) linking chemistry respectively. The chemical structure after each modification step was determined by attenuated total reflectance FTIR (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analyses. HA-grafted model soft contact lenses were characterized by improved surface wettability, antifouling and water retentive properties, while a decreasing trend in boundary friction was observed but only for the HA-grafted pHEMA-co-TRIS materials. Surface-tethering of rhPRG4 was found to effectively enhance the surface wettability and boundary lubricating properties of pHEMA-co-TRIS hydrogels only, whereas both rhPRG4-grafted pHEMA and pHEMA-co-TRIS materials exhibited lower protein sorption and dehydration rate. Overall, the surface immobilization processes followed herein did not alter the optical transparency of the model soft contact lenses or their in vitro compatibility with human corneal epithelial cells. Finally, there was evidence that HA and rhPRG4 synergistically interacted, further improving the contact lens properties. However, the degree of HA/rhPRG4 synergy was found to be dependent on the configuration of the formed HA/rhPRG4 complex as well as the composition of the substrate hydrogel material, with the noted improvement being more significant for the model silicone hydrogels. This is the first study to examine surface grafted full-length rhPRG4 and the effect of this modification on contact lens properties. Moreover, the study is the first to investigate the interactions between covalently tethered rhPRG4 and solutions containing HA. The results of this thesis demonstrate that HA and rhPRG4 are good candidates for the development of novel biomimetic surfaces, especially for silicone hydrogel contact lenses. The potential for using these compounds in synergy was also demonstrated, with wetting solutions of HA showing promise for modifying rhPRG4 modified materials to improve symptoms of discomfort. These naturally occurring ocular agents have the potential to improve the management of ocular dryness and discomfort, thus optimizing the overall soft contact lens performance. / Thesis / Doctor of Philosophy (PhD)