Blas Frangione, MD, Universidad de Buenos Aires Escuela de Medicina, PhD - US grants

The studies described in this proposal are designed to broaden our understanding of the genetic and structural basis of antibody specificity and idiotypes, the definition of immunoglobulin variants and the elucidation of the mechanism of amyloid fibril formation. The primary structure of the variable region of light and heavy chains of human monoclonal immunoglobulin M with antibody activity against self antigens and bacterial antigens will be determined in search of insights into the structural-idiotype-antigen specificity relationships and the genetic restrictions that appear to be present in autoantibody synthesis. The occurrence of variable region subgroups and complementarity-determining region antigens in monoclonal autoantibodies with defined specificity and normal immunoglobulins using antisera raised to synthetic framework residues fragments and complementarity determining region peptides will be studied. Our theory is that certain complementarity determining antigens and variable region subgroups are selectively associated with autoantibodies and that in spite of the heterogeneity of some autoantibody responses, the specificities of the binding sites and the idiotypes of autoantibodies are germline- gene encoded. The first clues to the discontinuous nature of gene coding for immunoglobulin chains, heavy chains in particular, emerged from serological and structural studies of Heavy Chain Disease proteins or immunoglobulin variants, which clearly indicated the existence of separate gene segments coding for different domains, a prediction confirmed by analysis of murine and human heavy chain gene structure. Studies of patients with these disorders and the proteins they produce are important in order to describe additional clinical syndromes associated with abnormal Ig synthesis and define their molecular defects. Studies of human AA amyloidosis and murine secondary amyloidosis due to chronic inflammation will be continued in order to confirm our hypothesis that amyloid fibrils are derived by degradation of a soluble precursor; that processing of the amyloid precursor is tissue specific, and that other factors, like amyloid enhancing factor, are involved in amyloidogenesis. This may in turn provide leads as to possible therapeutic approaches for the prevention of amyloid fibril formation and deposition.

Lewy Bodies (LBs) are distinctive intraneural cytoplasmic inclusions of unknown pathogenesis. They are found in all cases of Parkinson's Disease (PD), and diffuse Lewy body disease (DLBD) (also known as the Lewy body variant of Alzheimer's disease). In addition, LBs are occasionally found in a few other pathological processes. The composition of LBs by direct biochemical methods and their role in these diseases processes remains unknown. Recently we have shown that antibodies raised to purified amyloid extracted from familial amyloidosis, Finnish type (FAF) immunoreact with LBs. This immunoreactivity is specific, as it was absorbed by the purified amyloid proteins and is unaffected by other antigens such as ubiquitin, neurofilament proteins and tubulin which have been shown by immunohistochemical methods to be present in LBs. FAF amyloid has been found to be an aberrant degradation product, starting at position 173, of gelsolin, an actin modulating protein. The amino acid sequence shows heterogeneity at the N-terminus and at position 15, where asparagine substitutes for aspartic acid. At the DNA level, a guanine to adenine transition corresponding to nucleotide 654 of the human gelsolin has been found in all FAF patients tested so far. Commercially available anti- gelsolin antibodies raised to the carboxy-terminus of the molecule (away from the amyloidogenic region) do not immunostain LBs. This raises the possibility that LBs also contain an abnormal fragment of gelsolin that is closer to the amino terminal of the molecule. We plan: (1) To perform an immunohistochemical survey of DLBD, Alzheimer's disease, and Parkinson's disease cases with the anti-FAF antibody, as well as with antibodies to intact gelsolin, fragments and synthetic peptides of different regions of the gelsolin molecule, to evaluate patterns of immunoreactivity and epitope mapping; (2) To characterize the composition of the purified LBs preparation by using Western blotting and direct amino acid sequencing to sequence gelsolin and/or its fragments; (3) To investigate the biological activity of gelsolin isolated from DLBD, PD, AD, and control CSF to see if there are any abnormalities; (4) To clone the gelsolin gene from familial PD patients and screen for any variations; (5) To investigate cerebral spinal fluid from patients with PD, DLBD, other neurodegenerative disorder, controls and AD for the presence of any abnormal gelsolin fragments that could be used as a diagnostic test. Together these studies will expand knowledge about LBs and the role gelsolin plays in their formation.

Brain amyloidoses comprise a heterogeneous group of diseases that vary in clinical expression and the composition and distribution of insoluble amyloid fibrils. We believe that all types of cerebral amyloids involve altered degradation of an amyloid precursor protein, and that deposition occurs in patients who are genetically or otherwise defective in degrading these molecules. We put forward a tripartite division of the elements central to the issue of pathogenesis: the inherent chemical amyloidogenic potential of the precursor protein; the possibility of aberrant protein metabolism and/or genetic variants, and the existence of disease microenvironmental tissue factors that may facilitate the conversion of precursor proteins into their insoluble amyloid products. Recently we have shown that the amyloid fibrils of sporadic cerebral amyloid angiopathy and an autosomal dominant form of familial amyloid angiopathy in patients of Dutch origin (also designated Hereditary Cerebral Hemorrhage with Amyloidosis of Dutch Type) are similar to Alzheimer Disease amyloid beta-protein (Abeta). These findings indicate that Abeta deposition disorders form a spectrum of overlapping clinico-pathological conditions which range from patients with predominant vascular involvement to patients having a combination of vascular and parenchyma involvement in varying proportions, manifested clinically by stroke and dementia, respectively. We have also found that these disorders exhibit varying neuritic plaque-like structures, or "preamyloid lesions", suggesting that they represent an early stage of beta-protein deposition. Similar amyloid deposits are often encountered in a significant percent of neurologically asymptomatic aged individuals; hence it is important to clarify their relationship. We propose: 1) the biochemical and immunohistochemical analysis of preamyloid lesions obtained from Dutch patients with HCHWA and familial and sporadic forms of AD. 2) the characterization of amyloid protein and other components from familial and sporadic forms of AD. 3) performing similar studies in transgenic mice. This study is relevant for understanding the basic etiopathogenetic mechanisms associated with amyloid deposition in aging, amyloid angiopathy, and Alzheimer's Disease; it may be useful for diagnostic purposes at a pathological and clinical level and for therapeutic intervention.

DESCRIPTION (Investigator's Abstract): Hereditary cerebral hemorrhage with amyloidosis of Dutch type (HCHWA-D) is an autosomal dominant form of accelerated brain fibrillogenesis. It is characterized by extensive amyloid deposition in the small leptomeningeal arteries and cortical arterioles which leads to cerebral hemorrhage and early death. In most cases, there are also amyloid lesions in brain parenchyma resembling diffuse plaques. Distinct from Alzheimer's disease (AD) these deposits only occasionally are surrounded by dystrophic neurites and neurofibrillary tanges are absent. We have shown that the amyloid extracted from leptomeninges in HCHWA-D is a 39/40 residues peptide which is similar to the amyloid ( (A() obtained from AD, Down's syndrome and aged brains. This self-aggregating peptide is an internal proteolytic fragment of a larger membrane glycoprotein (PP, encoded by a single gene in chromosome 21. We detected a point mutation at codon 693 which causes a substitution of Glu for Gln at position 22 of A(. The mutation segregates with the disease and induces accelerated fibril formation in vitro. Our preliminary observations that A(17-42 (the "non-amyloidogenic" p3 fragment) might be a component of the diffuse plaques and that PS1 is present in both lesions in HCHWA-D, a disease not linked to chromosome 14, are puzzling. Our central interest lies in the apparent link between neurodegeneration and amyloidogenic proteins and in the determination of genetic and/or environmental factors that initiate and perpetuate amyloid formation. We postulate that HCHWA-D is a distinct entity that shares pathological similarities with AD and propose HCHWA-D as a model for cerebral amyloidosis in man. Better biochemical understanding of this pathological process and amyloidogenesis in humans and in animal models would facilitate therapeutic intervention in AD and aging. This proposal is directed to answer the following questions: Aim 1: Why diffuse plaques do not evolve to senile plaques in HCHWA-D? What is the biochemical composition of diffuse plaques and vascular amyloid deposits in HCHWA-D? What is the role of presenilins in amyloidogenesis? Aim 2: Can we inhibit, delay and/or revert the amyloidogenesis process designing amyloid analogs containing similar hydrophobicity but low propensity to adopt (-sheet conformation? Aim 3: Is the HCHWA-D model supportive of the hypothesis of the soluble origin of the A( deposited in brain lesions? Which is the role of the A( mutants in vascular localization, vascular sequestration, blood-to-brain and brain-to-blood transport in the in vivo perfusion model in guinea pigs?

This is a competing renewal of Merit Grant AR02594, which originally focused on the structure of immunoglubulins and their role in systemic amyloidosis. Although these investigations elucidated important structural data, the hypervariability of immunoglobulins and the lack of reliable animal models lead us to extend our studies to cerebral amyloidoses, and in particular the prion related disorders for which there are better model system. However, the central question we are addressing remains the same: What are the factors producing disease associated, abnormal protein conformation. The amyloidoses and several degenerative conditions are disorders of protein conformation, whereby a normal protein is structurally altered and is associated with the disease state. The prion diseases are the best defined human conformational diseases. Central to the etiology of prion disorders is the conversion of normal prion protein, PrP/C, into its infectious and pathogenic form, PrP/Sc. Current data indicate that PrP/c and PrP/Sc differ only in their conformation, with PrP/Sc have a greater beta-sheet content. However, identification of numerous "strains" of prions suggest the existence of multiple PrP/Sc conformations; alternatively, undefined co-factors may have a role in infectivity and strain specificity. We have postulated and demonstrated previously that molecular chaperones can bind and alter protein conformation leading to amyloidogenesis. In specific aim 1, we propose to identify PrP/Sc binding proteins and study their role in infectivity. and their strain-specific conformation properties. The existence of multiple conformations will be assessed by circular dichroism, FT-IR and proteinase K resistance of the isolated strains. The effect of PrP/Sc binding proteins on PrP/c conformation in vitro will be correlated with infectivity in vivo. In specific aim 2 we propose to construct synthetic peptides or mini chaperones capable of reversing pathologic protein conformation in order to modify PrP/Sc species in vitro and determine the effect of in vivo infectivity. Purified PrP/Sc, pretreated with mini-chaperone peptides specifically designed to bind and alter prior conformation will be assessed for alterations in infectivity and strain behavior. We shall test whether similar peptides can be used for in vivo imaging of PrP/Sc, utilizing [I/125]-labeling followed by autoradiography pr [19F]-labeling in conjunction with magnetic resonance spectroscopy. Our long-term goals are to define the structural features critical for the formation of PrP/Sc, which will have implications for the design rationale of effective pharmacological and diagnostic tools, as well as deepen our understanding of the basic mechanism(s) underlying conformational diseases.

DESCRIPTION (adapted from applicant's abstract):Since 1983 they have focused our efforts in the characterization of amyloid deposition in familial cerebral amyloid angiopathies of different origin and in defining their genetic defects. Hereditary cerebral hemorrhage with amyloidosis-Dutch type was the first familial form to be associated with mutations of the amyloid beta precursor protein gene. Subsequently, several genetic abnormalities in early onset familial Alzheimer's disease have been analyzed and transgenic mouse models carrying familial Alzheimer's mutants have been developed. However, neither the Dutch type nor animal models of Alzheimer's disease have clarified the relationship between amyloidogenesis and neuronal dysfunction. Vascular lesions and ischemic damage may play an important role in the pathogenesis of Alzheimer's disease since a combination of Alzheimer's disease and cerebral infarction is the second commonest type of dementia. Very recently we had the opportunity to analyze a new type of dementia - familial British dementia (FBD) - an autosomal dominant form of cerebrovascular amyloidosis with neurofibrillar degeneration leading to progressive dementia. We demonstrated that a stop codon mutation of a novel gene (BRI) localized on chromosome 13 gave rise to an elongated gene product. Degradation of the carboxyl-end of this abnormal precursor molecule releases a 34 amino acid amyloid peptide (ABri) found deposited in vascular and parenchymal plaques in patients with FBD. The principal investigators propose that ABri is the main cause of neurodegeneration and dementia in the British kindred and is an ideal model to study the relationship between amyloidogenesis, vascular pathology, and neurodegeneration. They plan: Aim I: (a) to biochemically and immunohistochemically define the composition of the amyloid deposits in vascular, perivascular, and parenchymal amyloid plaques; (b) to isolate and analyze soluble ABri species and its precursor(s) in biological fluids from FBD patients as well as in transfected cells. Dr Tjeu will also determine whether the disease is localized or systemic; (c) to precisely delineate the region(s) responsible for fibrillization via synthetic peptides of different length and composition. Aim II: to construct, develop and characterize a transgenic mouse model for FBD.