Diss. Linköping : Univ., 2001.

The plasma pentraxin proteins serum amyloid P component (SAP) and C-reactive protein (CRP) bind specifically to nuclear autoantigens. There is a blunted acute phase response of human CRP in systemic lupus erythematosus (SLE), and of mouse SAP in NZB/W murine SLE. SAP deficiency in (129/Sv X C57BL/6)F2 mice is associated with spontaneous antinuclear autoimmunity. The pentraxins may thus function in preventing autoimmunity. Pure line C57BL/6 SAP knockout mice, studied here for the first time, spontaneously developed broad spectrum anti-nuclear autoimmunity resembling human SLE, and, females in particular had proliferative immune complex glomerulonephritis but without proteinuria or renal failure. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP-/- than in wild-type mice. Extrinsic chromatin was catabolised predominantly in hepatocytes, Kupffer's cells and renal parenchymal cells. Plasma clearance of long chromatin and core particles was marginally slower in SAP-/- mice, with significantly greater splenic uptake of nucleosome core particles, which may have immunological significance. SAP bound to apoptotic cells but had no effect on their phagocytosis in mice in vivo, or by human macrophages in vitro. CRP also bound to apoptotic cells but did not compete with SAP, indicating recognition of different ligands. In contrast, to the C57BL/6 strain, pure line 129/Sv SAP knockout mice did not produce autoantibodies. Formation of antinuclear autoantibodies is thus markedly strain dependent. Interestingly, transgenic expression of human SAP in the C57BL/6 SAP knockouts did not abrogate the autoimmune phenotype. Transgenic reconstitution of the knockouts with mouse SAP is currently in progress to confirm that autoimmunity is caused by SAP deficiency and not by 129/Sv genes transferred into the C57BL/6 background during homologous recombination, or by disruption of loci associated with murine SLE that are close to the mouse SAP gene.

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Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. Although the etiology of the disease is yet to be fully elucidated, pathological hallmarks have been consistently described, including the accumulation of amyloid plaques, dysfunctional ionic homeostasis, synaptic disruption and neurodegeneration. The amyloid hypothesis postulates that aberrant production of amyloid-β (Aβ) proteins, which have a high propensity to aggregate, lies at the center of the pathological mechanism of AD. In particular, soluble oligomeric Aβ structures have been identified as primary toxic species. The interaction of these structures with several cellular targets, including ion channels such as nicotinic acetylcholine receptors (nAChR) and voltage operated Ca²⁺ channels (VOCC), has also been implicated in Aβ toxicity and AD. The aim of this thesis is to investigate how the acute and chronic actions of Aβ in vitro are affected by nicotinic ligands. Acute application of Aβ₁₋₄₂ to fluo-3-loaded PC12 cells potentiated Ca²₊ increases evoked by stimulation of nAChR and VOCC, while chronic application reduced redox potential, disrupted membrane integrity and initiated apoptosis in PC12 cells. In addition to mimicking the toxic responses of PC12 cells, Aβ₁₋₄₂ also reduced neurite outgrowth and synaptogenesis in rat primary cortical neurons. All actions of Aβ were prevented by inhibitors of Aβ₁₋₄₂ oligomerisation, including the hexapeptide KLVFFA. Neuroprotection afforded by (+)-nicotine also occurred via inhibition of Aβ₁₋₄₂ oligomerisation, rather than by a receptor-mediated mechanism. No other pharmacological approaches, including application of two novel ligands selective for α7 nAChR: the partial agonist SSR180711 and antagonist α-conotoxinArIB[V11L,V16D], characterized herein, protected against Aβ₁₋₄₂ toxicity. While inhibiting oligomerisation prevented the actions of Aβ₁₋₄₂, enhanced oligomerisation evoked amplified toxic responses. However, the potentiation of Ca²⁺ signalling diminished following enhanced oligomerisation. This, coupled with a lack of VOCC-involvement in Aβ toxicity and the differential actions of truncated Aβ peptides on toxicity and Ca²⁺ signaling, indicates that the acute disruption of Ca²⁺ signaling by Aβ does not underpin the chronic toxic effects of Aβ.

Most proteins must fold into their native conformations to fulfil their biological functions. Failure of proteins to fold leads to cell pathology and a broad range of human diseases referred to as protein misfolding disease, e.g., Alzheimer’s disease, Parkinson’s disease, and type II diabetes. More than 40 proteins are known to be connected with misfolding diseases. These proteins share no sequence homology but all assemble into cross-b sheet containing insoluble fibrillar aggregates. Despite the pathological conditions that these proteins can induce, living organisms can take advantage of the inherent ability of these proteins to form such structures and to generate novel and diverse biological function, the functional amyloid.  This thesis examines different aspects of cross-b sheet containing aggregates. The first paper describes the humoral response to aggregated structures of insulin and the astrocytical biomarker S100B in patients suffering from Parkinson’s disease. We show that the patients have an increased immunreactivity towards insulin and S100B in Parkinson’s disease patients compared to a control group.  The second part of this work focuses on a functional amyloid. HAMLET (human a-lactalbumin made lethal for tumour cells) is a complex of a-lactalbumin and oleic acid, which kills tumour cells but not healthy differentiated cells. We wish to expand the concept of HAMLET to a structurally related protein and therefore create and characterize a complex of equine lysozyme and oleic acid (Paper II). We chose equine lysozyme because both proteins (equine lysozyme and a-lactalbumin) share common ancestors and are spatially related. The newly designed complex was named ELOA, for equine lysozyme with oleic acid. ELOA represents a functional oligomer due to its multimeric state and its ability to bind amyloid specific dyes. In the third paper, we investigate the interaction of the cytotoxic ELOA with live cells in real time to find a mechanistic model (Paper III).  It is known that HAMLET is not only tumouricidal but is also toxic towards many bacteria. Therefore in the last part of the thesis, we investigated the effects of ELOA on different bacterial strains and focused on its interplay Streptococcus pneumoniae (Paper IV).  These studies have added significantly to many aspects of protein folding and misfolding from its involvement in Parkinson’s disease to the newly gained functions and structural aspects of de novo produced ELOA.

Chronic inflammation in response to Aß peptide deposits is a pathological hallmark of Alzheimer's disease (AD). The inflammatory environment includes populations of reactive and proliferating microglia and astrocytes and perturbed vasculature. However, the association between activated glial cells and cerebrovascular dysfunction remain largely unknown. This study has used Aß1-42 intrahippocampal injection as an animal model of inflamed AD brain to characterize mechanisms of glial-vasculature responses as a basis for chronic inflammation. Preliminary findings suggested Aß1-42-injected brain demonstrated vascular remodeling including evidence for formation of new blood vessels (angiogenesis). This result led to study of the effects of the anti-angiogenic/anti-inflammatory compound, thalidomide on activated glial cells and perturbations in the vasculature in an Aß1-42 peptide-injected rat model. First, Aß1-42 injection was found to cause perturbations in vasculature including new blood vessel formation and increased BBB leakiness. Second, thalidomide decreased the vascular perturbations and the glial reactivity and conferred neuroprotection. Overall, these results suggest that altered cerebral vasculature is integral to the overall inflammatory response induced by peptide. Experiments then examined the level of parenchymal plasma proteins in brain tissue from AD and nondemented (ND) individuals. AD, but not ND, brain tissue demonstrated high levels of fibrinogen immunoreactivity (ir). Aß1_42 injection into the rat hippocampus increased the level of parenchymal fibrinogen, which was reduced by treatment with the defibrinogenating agent, ancrod. In addition, ancrod also attenuated microglial activation and prevented neuronal injury. Overall, these results demonstrate that extravasation of blood protein and a leaky BBB are important in promoting and amplifying inflammatory responses and causing neuronal damage in inflamed AD brain. Microglial chemotactic responses to VEGF (vascular endothelial growth factor) receptor Flt-1 were next studied. Treatment with a monoclonal antibody to Flt-1 (anti-Flt-1 Ab) in the peptide-injected hippocampus diminished microglial reactivity and provided neuroprotection. Secondly, anti-Flt-1 Ab inhibited the AI3142-induced migration of human microglia. These results suggest critical functional roles for Flt-1 in mediating microglial chemotaxis and inflammatory responses in AD brain. The overall conclusion from my work is that AP deposits induce microglial reactivity which subsequently causes vascular remodeling resulting in an amplified inflammatory microenvironment which is damaging to bystander neurons.

The work described in this dissertation details the development of a biomimetic materials for use in sensors and therapeutics, based on new advances in material science. The sensors developed herein target neurodegenerative diseases. Two of the diseases, the transmissible spongiform encephalopathies (TSEs) and Alzheimer’s disease (AD), are diseases associated with the abnormal folding of a protein, thus detecting the disease is dependent upon developing structure specific sensor technologies. Both sensors developed in this work take advantage of the unique optical properties associated with nanoscale metal particles, however they use different types of spectroscopies for optical detection of the presence of the disease associated abnormal protein, and different types of recognition elements that bring the disease associated proteins close to the nanoscale metal particles. In the case of TSEs, the recognition element was a commercially available antibody. In the case of AD, the recognition element was a molecular scale self-assembled surface. A therapeutic for AD was developed based on the molecular scale materials developed for the AD biosensor. Mathematical models were developed that facilitated the rational design of the biosensors described in this work that could also be used in future biosensor development.

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