Search

A number of forms of congenital muscular dystrophy (CMD) have now been identified that involve defects in the glycosylation of dystroglycan with O-mannosyl-linked glycans. Unlike many neurologic disorders, altered glycosylation in CMDs does not cause aberrant protein aggregation or loss of expression. Instead, defects in glycosylation inhibit the binding of extracellular matrix proteins such as laminin to dystroglycan on neuronal and muscle cell membranes, thereby causing cellular pathology. Overexpression of one gene implicated in CMD, LARGE, can increase dystroglycan glycosylation and restore its function in cells taken from CMD patients. Thus, stimulating protein glycosylation may also be an avenue to treatment of these disorders. [ABSTRACT FROM AUTHOR]

Increased glycation is associated with aging and complications of diabetes. So it is not surprising that glycation has been a subject of intensive study. Most emphasis is on long-lived proteins, mostly structural, because they are exposed to the sugars for a longer time than enzymes and other nonstructural proteins in most tissues. However, glycation is not specific and is not restricted to structural proteins. Besides leading to the functional impairment of modified proteins, glycation was shown to produce significant structural alterations, resulting in modified proteins with properties similar to those of "molten-globule" intermediates of protein folding and unfolding pathways. Evidence supporting the role of nonenzymic post-translational modification of lens proteins in cataract is overviewed. [ABSTRACT FROM AUTHOR]

Several hypotheses have been advanced in order to explain the molecular mechanisms by which expanded polyglutamine (polyQ) triggers neurodegeneration. In this chapter, we discuss the experimental evidence supporting the leading hypotheses in the field. In particular, we focus on the mechanisms by which abnormal protein folding, oligomerization, and aggregation may impair neuronal function and survival in the dominant spinocerebellar ataxias (SCAs) caused by polyQ expansion. [ABSTRACT FROM AUTHOR]

Spinal and bulbar muscular atrophy (SBMA) is an adult onset neurodegenerative disease that affects men beginning in the fourth to fifth decade of life. The disease results from the expansion of a CAG trinucleotide repeat in the first exon of the androgen receptor gene. Symptoms of SBMA include weakness and atrophy of the proximal limb and bulbar muscles as well as partial androgen insensitivity. Cellular markers of disease include the loss of spinal motor neurons and the formation of nuclear aggregates of mutant androgen receptor protein (NII). The mechanism for pathogenesis in SBMA is unknown, and there is currently no treatment for the disease. New transgenic mouse, fly, and cell models of SBMA have provided important insights into the role of testosterone binding as a critical initiator of disease. An understanding of androgen receptor (AR) metabolism upon ligand binding is likely to provide new insights into the upstream events in SBMA pathogenesis. Moreover, the creation of novel models of disease is providing insights into abnormal neuronal responses to expanded AR accumulation. Here we discuss these ideas in the context of both basic AR biology and novel therapeutic development for SBMA. [ABSTRACT FROM AUTHOR]

Missense mutations and gene multiplication in the α-synuclein gene cause inherited forms of Parkinson's disease (PD), and deposition of amyloid fibrils composed of wild-type α-synuclein in Lewy bodies and Lewy neurites is a pathologic hallmark of sporadic PD and other related neurodegenerative diseases such as dementia with Lewy bodies. These findings, along with the studies in animal models, strongly suggest that misfolding and aggregation of α-synuclein is a critical component in the pathogenesis of these disorders. Though the physiologic function of α-synuclein has not been completely defined, there has been a body of evidence supporting its roles in synaptic transmission, synaptic vesicle biogenesis, and lipid transport and metabolism. More importantly, based on the lack of neurodegenerative phenotypes in synuclein knockout animals, it has been suggested that loss of normal function of this protein might not be the direct cause of PD. α-Synuclein forms various types of nonfibrillar and fibrillar aggregates. PD-linked mutations accelerate the aggregation process in one or more steps in the fibrillation. Although some oligomeric aggregates seem to be toxic to cell, leading to functional abnormalities such as proteasomal and lysosomal dysfunctions, mitochondrial deficits, and Golgi fragmentation and transport defects, cells have defense mechanisms against these potentially toxic oligomeric aggregates. The free oligomers are transported to and deposited in the pericentriolar region by the microtubule system, resulting in the sequestration of toxic aggregates. After deposition, the transition from oligomers to fibrils occurs, forming Lewy body-like inclusion bodies and perhaps transforming toxic species into inert aggregates. Moreover, we have also shown that cells can degrade the preformed oligomers by autophagy. Elucidation of the mechanisms of the cellular aggregation process and the handling of preformed toxic aggregates should greatly enhance our understanding of the disease and provide rational targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Glutamine/asparagine-rich sequences are physiologically used for prion-based propagation of yeast phenotype mediated by Ure2 and Sup35, which accommodate yeast cells to changes of the nutritional condition. In addition, the repeat sequences are critically involved in pathogenesis of polyglutamine diseases. In this chapter, I review general outlines of the physiologic and pathologic roles of glutamine-rich and asparagine-rich sequences in proteins. [ABSTRACT FROM AUTHOR]

At first sight, the lens of the eye would appear to be an ideal environment for amyloid fibril formation. The protein concentration is the highest of any tissue in the body, the proteins are very long-lived, present in slightly acidic conditions and subjected over time to extensive truncation and post-translational modification. In addition, it has been demonstrated that lens crystallins can readily be induced to form amyloid fibrils in vitro. The situation may be further exacerbated after the onset of age-related nuclear cataract, which is characterized by massive oxidation of cysteine and methionine residues, accompanied by protein unfolding. Despite this, there is as yet no evidence for amyloid fibril formation in either the aged or the cataract human lens. Paradoxically, the reason may have to do with the supramolecular ordered β-sheet array that crystallins adopt once they are packed into mature fiber cells. This extended matrix in normal lenses displays some of the classic features normally associated with amyloid, for example, staining with Congo red and thioflavine T. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is caused by mutation(s) affecting the dystrophin gene that codes for a cytoskeletal protein. Dystrophin interacts with cytoskeletal actin filaments and with a complex of transmembrane glycoproteins. This assembly disintegrates when dystrophin is absent. The purpose of this chapter is to review the structural and functional disorders observed in dystrophin-deficient muscles in order to understand their role and evaluate their importance in the pathologic processes that eventually lead to fiber necrosis. A preliminary brief presentation outlines the main facts of DMD and its genetics in humans. As an enormous wealth of data have been obtained from studies of the mdx mouse, a natural mutant lacking dystrophin, an extensive description and analysis of the dystrophic phenotype of this animal model of DMD is presented. The review covers the fields of histology, regeneration, mechanics, electrophysiology, intracellular calcium homeostasis, protease activation, protein over/down expression, inflammatory response and microcirculation in mdx muscles. An attempt is made to distinguish between initial pathologic processes, thought to be specific to the lack of dystrophin, from downstream events. The loss of the mechanical strength of the fiber plasma membrane resulting from the lack of dystrophin and of its associated protein appears to be the primary defect responsible for initiating the pathologic cascade of muscle dystrophy. Consequences of this situation for the development of compensatory therapies is discussed. [ABSTRACT FROM AUTHOR]

The neuromuscular disorders are a heterogeneous group of genetic diseases, causing a progressive loss of the motor ability. In the past decade, mutations in several genes have been identified, resulting in the deficiency or loss of function of different important proteins. Complementary biochemical and immunohistologic analyses have localized these proteins in several compartments of the muscle fiber: in the sarcolemmal muscle membrane (dystrophin, sarcoglycans, dysferlin, caveolin 3), extracellular matrix (α2-laminin, collagen VI), in the sarcomere (telethonin, myotilin, titin), in the muscle cytosol (calpain 3, FRPR, TRIM32), and in the nucleus [emerin, lamin A/C, survival motor neuron gene (SMN) protein]. In the muscular dystrophies group, the allelic X-linked Duchenne and Becker forms are caused by mutations in the dystrophin gene. Among the Limb-girdle forms, seven autosomal dominant [limb-girdle muscular dystrophy (LGMD)1A to LGMD1G] and twelve autosomal recessive (LGMD2A-2L) are already known. The genes for the LGMD1 types were mapped respectively at 5q22-q34 (myotilin), 1q11-21 (lamin A/C), 3p25 (caveolin-3), 6q23, 5q31, 7q, and 4p21. Among the LGMD2 forms, the four clinically more severe types, the sarcoglycanopathies (LGMD2C-2E, SGpathies), mapped at 17q21, 4q12, 13q12, and 5q33, encode respectively for α-sarcoglycan (α-SG), β-SG, γ-SG, and δ-SG, which are glycoproteins of the sarcoglycan subcomplex of the dystrophin-glycoprotein complex (DGC). The other eight adult forms are LGMD2A at 15q (calpain 3), LGMD2B, at 2p31 (dysferlin), LGMD2G at 17q11-12 (telethonin), LGMD 2H at 9q31-33 (TRIM32), LGMD2I at 19q13.3 (Fukutin-related protein; -FKRP), and LGMD2J at 2q24 (titin), LGMD2K at 9q-4 (POMT1) and LGMD2L at 9q31 (Fukutin). Among the congenital forms, the congenital muscular dystrophy (CMD)1A with α2-laminin deficiency (at 6q2) is the most common accounting for about 50% of the cases. Protein studies are of utmost importance for the elucidation of the pathophysiology of each genetic disorder involved. It also can be used for the differential diagnosis and to direct the search for gene mutations, mainly because, in addition to genetic heterogeneity, most of the known genes are very large and present a wide variability of pathogenic mutations. Genotype-phenotype correlation through the analysis of the effect of different mutations on protein expression and on phenotypic variability contributes to the understanding of gene function. [ABSTRACT FROM AUTHOR]

Protein aggregation in muscle fibers may be a nonspecific phenomenon such as occurring in cores or ragged red fibers. However, it may also be a disease-specific and disease-significant phenomenon constituting protein aggregate myopathies (PAMs). These may be divided into two classes: The first one is marked by impaired extralysosomal degradation of proteins, catabolic PAM, encompassing desmin-related myopathies. Mutant proteins, that is, desmin, myotilin, or α-B crystallin, defy protein degradation, aggregate and associate with other proteins within muscle fibers, hence marking desminopathies, myotilinopathies, and α-B crystallinopathies. A second class of PAM encompasses those apparently associated with developmental errors though, again, based on mutations in genes for myofibrillar proteins, foremost sarcomeric actin and myosin. Here, actinopathies and myosinopathies often occur early in childhood while catabolic PAMs are largely of adult or even late onset. The common principle of these PAMs is that immunohistochemical identification of certain proteins resulted in subsequent molecular analysis of respective genes, identification of mutations, and demonstration of mutant proteins as important components of these protein aggregates. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein p53 is a key control in the cell cycle and plays a crucial role in the prevention of cancer development. It is mutated in approximately half of all human cancers and has, therefore, become an important target for the development of novel cancer therapies. Here, we review the structure of the protein, the effects of mutation and how they may be reversed. p53 has a highly complex domain organization consisting of structured regions combined with largely unstructured domains. Most cancer-associated mutations are located in the DNA-binding core domain of the protein. The molecular basis for the detrimental effect of these mutations has been elucidated by structural and biophysical studies. Whereas some mutations affect residues that make direct contact with target DNA, others induce structural perturbations that reduce the thermodynamic stability of the protein. Because p53 core domain is only marginally stable above body temperature, many cancer mutations not only induce local conformational changes but also cause global unfolding of the core domain under physiologic conditions. Novel therapeutic strategies aim, therefore, to develop chemical chaperones that help refold p53 mutants to their correct native structure. Valuable lessons can be learned from studies on so-called second-site suppressor mutations that reverse the effects of cancer mutations. These may provide a basis for the rational design of novel therapeutics. [ABSTRACT FROM AUTHOR]

The structural and biochemical properties of human copper-zinc superoxide dismutase (SOD1) have been studied for more than 30 years. Although most enzymes give up their secrets over such a long period, SOD1 has continued to be the subject of intense investigation, most recently due to the discovery of its linkage to an inherited form of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS; motor neuron disease, Lou Gehrig's disease). Approximately 100 mutations in SOD1 are now known to give rise to ALS, most of which are single amino acid substitutions. Accumulating evidence strongly suggests that the majority of these lesions lead to SOD1 aggregation, and it appears that this nonnative SOD1 self-association is somehow injurious to motor neurons. However, the detailed molecular mechanism(s) through which these SOD1 mutants exert their toxic effects remain largely undefined. The information contained in this chapter is intended to provide a synopsis of what is currently known and/or hypothesized about the molecular basis for SOD1-linked ALS, with a strong emphasis on the biophysical and structural properties of the enzyme. Enhanced understanding of the structural basis for pathogenic SOD1 misfolding and self-association is likely to be a prerequisite for the development of therapeutic avenues of this progressive neurodegenerative disease. [ABSTRACT FROM AUTHOR]

The diverse members of the serpin family of protease inhibitors can each typically undergo a profound change in conformation. Aberrations of this conformational transition frequently occur and lead to a range of diseases, including emphysema, cirrhosis, thrombosis, and dementia, each of the diseases reflecting the functions and site of synthesis of the individual serpins, as illustrated here with α1-antitrypsin, antithrombin, and neuroserpin. The molecular pathology of these aberrations has been studied in crystallographic detail. So the serpins now provide an archetypal example of the diversity of changes that underlie the conformational diseases. The studies show, in structural detail, how a range of consequences can arise from even a single conformational aberration. The mutant serpins can exceptionally align to give highly ordered amyloid-like fibrils, but the most studied manifestation of their structural instability is the formation of betalinked polymers that aggregate as endoplasmic inclusion bodies. However, the same mutations that cause the intracellular polymerization of a serpin can also give rise, in the same serpin, to monomeric transitions and to the formation of soluble dimers and oligomers. Each of these forms has potentially toxic effects, and the different proportions and consequences of each account for the variability of the associated diseases. [ABSTRACT FROM AUTHOR]

Lysozyme is perhaps the protein whose structure, stability, and folding behavior has been studied most widely over many years to define general principles underlying these complex phenomena. The relatively recent and unexpected observation that lysozyme is one of the 20 or so proteins whose conversion into amyloid deposits is associated with debilitating medical conditions has enabled these studies to be extended to probe the nature and origins of the misfolding events that underlie this type of disease. In this chapter, we summarize the results of these investigations and discuss both the specific mechanism through which lysozyme forms amyloid fibrils and the manner in which this process can be inhibited for potential therapeutic benefit. In addition, we discuss briefly how studies of lysozyme have provided new insights into links between normal and aberrant folding and into the way living systems avoid the consequences of the inherent tendency of polypeptides to convert into intractable and frequently toxic aggregates. [ABSTRACT FROM AUTHOR]

Transthyretin is a normal serum protein that carries the secondary thyroid hormone thyroxine and retinol binding protein when it is loaded with retinol. It is synthesized primarily in the liver but there is also significant production in the choroid plexus and the retina. Both message and protein are found in the kidney but that site does not appear to contribute to the serum level in any meaningful way. The protein is a homotetramer composed of the 14-kDa monomer with the thyroxine binding sites in the central groove. The structure is highly conserved particularly in the regions responsible for ligand binding, suggesting that its carrier function has been retained over the millennia. More than 80 mutations at 55 different positions in the gene encoding the protein are the primary cause of a set of human disorders collectively known as the familial amyloidotic polyneuropathies and cardiomyopathies. In these diseases, the soluble protein becomes insoluble under physiologic conditions resulting in functional compromise in the organs in which the protein is deposited. Peripheral nerves, heart, kidneys, gastrointestinal tract, and the leptomeninges have all been described as sites of deposition. It is possible that particular syndromes are associated with particular sets of mutations but, because of the rarity of some of the mutations, it is uncertain if the relationship between any mutation and any clinical syndrome is absolute. Further, it is also clear that the wild-type protein can deposit in tissues with subsequent dysfunction, particularly in the heart and carpal tunnel. Biophysical studies in vitro indicate that the process leading from soluble tetramer to insoluble aggregates involves monomer release, misfolding, oligomerization, and extension and lateral aggregation apparently as a downhill polymerization to a more stable lower energy state. How this is modulated in vivo is not known, although accessory molecules appear to be involved in the process. The early, albeit incomplete, understanding of the processes have led to potential therapies directed at stabilizing the tetramer or interfering with the interaction with other molecules as well as replacing the offending gene by liver transplantation. [ABSTRACT FROM AUTHOR]

Amyloid-related diseases show marked extracellular deposition of nonbranching protein fibrils, called amyloid, in various soft organs. The mechanisms by which soluble amyloid precursor proteins transform into amyloid fibrils remain mainly obscure. Here we discuss data derived from alveolar hydatid cyst-infected (AHC) mouse model of inflammation-associated amyloid A (AA) amyloidosis and concepts related to the potential roles of oxidative stress-related factors and metabolism of monocytoid cell-associated serum amyloid A (SAA) precursor protein in AA fibril formation in vivo. Evidence shows that AA fibrils are generated intracellularly within the activated host monocytoid cells, which prior to and during AA fibril formation generate, among other oxidative stress-related derivatives, 4-hydroxynonenal, a lipid peroxidation product. We suggest that release of the AA fibrils formed intracellularly, either by exocytosis or cell death, could act as the "seed" for the nucleation-dependent expansion of extracellular AA fibril deposition. [ABSTRACT FROM AUTHOR]

β2-Microglobulin (β2m) is the noncovalently bound component of the class I major histocompatibility complex (MHC) and is one of more than 20 proteins that are known to cause human amyloid disease. β2m is degraded and excreted by the kidney as part of its normal catabolic cycle. In patients suffering renal failure, therefore, the principal mechanism of β2m degradation is abrogated, with the consequence that the concentration of β2m in the serum increases by up to 60-fold, which leads to the association of freely circulating β2m into insoluble amyloid fibrils, which typically accumulate in the musculoskeletal system, causing dialysis-related amyloidosis (DRA). The unique environment of the synovial joint may also be an important feature in stimulating β2m deposition in a fibrillar form, although the factors that play the key role in the development and deposition of β2m amyloid in this compartment remain unclear. In this chapter, we outline the events involved in the development of DRA. We discuss biophysical and biochemical studies of β2m fibrillogenesis in vitro and summarize the insights that these studies have provided into the structural molecular mechanism of its self-association into amyloid fibrils. We then discuss the role of different biological factors in influencing amyloid formation in vitro and in vivo, highlighting key questions about the mechanism of aggregation of β2m into amyloid fibrils that have arisen from these studies. Finally we discuss routes forward in the search for potential new therapies for DRA patients. [ABSTRACT FROM AUTHOR]

Deposition of amyloid in pancreatic islets is a feature of type 2 diabetes in man but the causal factors are unknown. Progressive amyloidosis results in loss of insulin-producing cells and increased severity of disease. The fibril component is a 37-amino-acid peptide, islet amyloid polypeptide (IAPP; amylin), which is a normal cellular peptide, cosecreted with insulin; the species-specific amino-acid sequence of IAPP is unaltered in diabetes. Peptide refolding in islet perivascular spaces to form β-sheets could be related to production of fragments of the precursor peptide, proIAPP. Islet amyloidosis is not a causal factor for hyperglycemia in man but contributes to onset of diabetes in non-human primates and transgenic rodent models. Because islet amyloidosis cannot be detected in vivo, clinical development of inhibitors is likely to be difficult. [ABSTRACT FROM AUTHOR]

Light-chain amyloidosis (AL) is characterized by the clonal expansion of plasma B cells that secrete large amounts of monoclonal immunoglobulin light chains. The free light chains circulate in serum and form amyloid fibrils on vital organs such as the kidney, heart, and liver causing organ failure and eventually death. Multiple myeloma (MM) is another type of B-cell malignancy. MM results in bone lesions, hypercalcemia, renal failure, and anemia. Free light chains from MM patients are non-amyloidogenic. There is a small subset of MM patients (10-15%) that also develop light-chain amyloidosis complications. Amyloid fibrils are derived from the N-terminal region of the light-chain variable domain (VL). Due to the antigen-driven selection process, there is a large degree of mutational variability, thus each patient has a unique VL sequence. In some cases, the organ involvement has some correlation between VL subtype and germ-line donor sequence gene. To understand the amyloidogenicity of AL proteins, their thermodynamic properties have been compared with non-amyloidogenic MM proteins. Generally, AL proteins have lower thermodynamic stability than MM proteins. Current and future research is focusing on understanding the mutational diversity and organ tropism associated with AL, as well as understanding which species along the amyloid fibril formation pathway causes cellular toxicity. The current treatment for AL targets the plasma cell clone. Chemotherapy and peripheral stem cell transplantation are commonly used. Future therapies to treat this disease could involve small molecules that stabilize the folded state and inhibit amyloid fibril formation and molecules that bind to amyloid fibrils, destabilize the fibrils, thus reducing the amyloid burden. In this chapter, we discuss the structure, truncations, mutational diversity, and organ tropism of immunoglobulin light chains, thermodynamics, and fibril formation studies using AL and MM proteins and the current treatment options and also discuss directions in the study and treatment of AL. [ABSTRACT FROM AUTHOR]

In the yeast Saccharomyces cerevisiae, two genetic elements, [PSI+] and [URE3], were discovered nearly 40 years ago (Cox, 1965; Lacroute, 1971). These traits do not obey Mendel's laws. They were first considered to be due to a nonchromosomal nucleic acid. However, [PSI+] and [URE3] behavior differs significantly from that of DNA plasmids, RNA viruses, the mitochondrial genome, or RNA replicons. In addition, the genes encoding these traits are located in the nucleus of yeast cells (Schoun and Lacroute, 1969; Cox et al., 1988). The very unusual properties of [PSI+] and [URE3] led only 10 years ago to extend the prion hypothesis to the yeast Saccharomyces cerevisiae (Wickner, 1994). The term prion is meant for infectious protein that can lose for unknown reasons its normal function and that converts the functional polypeptide into a nonfunctional form. This chapter defines the genetic criteria that define a prion in yeast and relates the discovery of S. cerevisiae prions. The structural features of the soluble and insoluble forms of S. cerevisiae prions are detailed, and the mechanistic models for aggregation are presented. Finally, the mechanisms of maintenance and propagation of yeast prions, in particular the role played by molecular chaperones and the potential role of yeast prions, are described. [ABSTRACT FROM AUTHOR]

The discoveries of prion disease transmission in mammals and a non-Mendelian type of inheritance in yeast has led to the establishment of a new concept in biology, "the prion hypothesis." This hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner by recruiting the cellular isoform of the same protein, and, therefore, acts either as transmissible agent of disease (in mammals) or as heritable determinant of phenotype (in yeast and fungus). While the prion biology of yeast and fungus supports this idea strongly, the direct proof of the "protein-only" hypothesis in mammals, the generation of PrPScin vitro, de novo from noninfectious PrP, has been difficult to achieve despite many years of effort. The current review discusses potential strategies for generation of prion infectivity de novo and summarizes our current knowledge about biophysical mechanisms of prion conversion. [ABSTRACT FROM AUTHOR]

Prion diseases are invariably fatal neurodegenerative disorders affecting humans and many other mammals. Here we discuss the current understanding of prion biology and the pathogenesis of this group of illnesses. We introduce several aspects of prion biology, from the primary structure of the cellular form of the prion protein (PrPC) to the conformational changes that occur during the conversion to the pathologic form, PrPSc. Moreover, we provide an overview of the various prion diseases. We then conclude with the recent discovery of mammalian synthetic prions and the implications that such findings may have to the future of prion research. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a multifactorial movement disorder in which both genetic and especially environmental factors play important roles. Substantial evidence implicates the aggregation of α-synuclein, an abundant and conservative presynaptic brain protein with unknown function, as a critical factor in PD. Rare familial cases of PD are associated with the mutations A30P (Ala to Pro substitution at position 30), E46K (Glu to Lys substitution at position 46), and A53T (Ala to Thr substitution at position 53) in α-synuclein. The primary structure of α-synuclein is characterized by several unusual motifs, and this protein was shown to have two closely related homologues, β-synuclein and γ-synuclein. Under the physiologic conditions in vitro, α-synuclein is characterized by the lack of rigid well-defined 3-D structure (i.e., it belongs to the class of natively unfolded proteins). Intriguingly, α-synuclein is known to possess remarkable conformational plasticity. The structure of this protein depends dramatically on the environment, and a number of absolutely unrelated conformations have been observed, including a partially folded intermediate that is a key intermediate in aggregation and fibrillation, several oligomeric species, and fibrillar and amorphous aggregates. A number of factors that either accelerate or inhibit the rate of fibrillation in vitro have been described. Accelerators include environmental factors such as certain pesticides and metals, molecular crowding, and various natural and synthetic charged polymers. Inhibitors include high concentrations of stabilizers such as trimethylamine N-oxide (TMAO), certain catechols, rifampicin, baicalein, acidic lipid vesicles, and protein homologues (β- and γ-synucleins). Oxidation of the four methionine residues in α-synuclein leads to the abolishment of fibrillation, as does the nitration of tyrosine residues. There is a strong correlation between the conformation of α-synuclein (induced by various factors) and its rate of fibrillation. The aggregation process appears to be branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous deposits. The molecular basis of Parkinson's disease appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. [ABSTRACT FROM AUTHOR]

During the past 30 years, a number of different hypotheses on cause of neuronal degeneration and dysfunction in Parkinson's disease have been intensively investigated. Roles have been postulated for oxidative stress, excitotoxcity, nitric oxide, mitochondrial dysfunction, and inflammation. The possibility of an environmental cause was highlighted by the discovery of a simple molecule known as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that is selectively toxic to the same cells in the brain that die in Parkinson's disease. Yet the most recent hypothesis to come under scrutiny did not emerge from any of this previous work, but rather from a rare form of genetic parkinsonism known as PARK 1. This form of autosomal dominant parkinsonism is caused by mutations in the gene that encodes for the protein α-synuclein. Unexpectedly, this protein has been found to accumulate in nerve cells and their processes in all patients with Parkinson's disease, raising the possibility that abnormal protein folding and aggregation represent a fundamental feature of the disease. These observations have already ushered in a new era of research on the disease and stimulated novel strategies directed toward disease modification by providing new therapeutic targets for drug development. Only time will tell if this most recent chapter in the search for the molecular basis of neurodegeneration in Parkinson's disease will be one that holds the key to understanding this complex disorder and the highly characteristic pattern of selective vulnerability exhibited by the neuronal populations it affects. But an exciting body of accumulating scientific evidence is pointing in that direction. [ABSTRACT FROM AUTHOR]

Two of the characteristic pathologic features present in the brains of Alzheimer's disease (AD) patients are the deposition of aggregated amyloid-β peptide (Aβ) and high levels of oxidative stress. Both these phenomena can be explained by Aβ's interactions with metal ions. When Aβ coordinates Zn, Cu, and Fe, the peptide aggregates. If the metals are redox active such as Cu and Fe, then reactive oxygen species (ROS) are generated. The generation of ROS has been implicated in the toxic mechanism of Aβ. A class of metal-protein attenuating compounds (MPACs) that are capable of inhibiting Aβ-metal interactions have been developed. The prototypic MPAC, clioquinol, has shown efficacy in cell and animal models of AD and promising results in a small-scale phase IIa clinical trial. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause for cognitive decline in the elderly. The disease results in relentlessly progressive decline in intellectual function and gradual loss of activities of daily living. Ninety percent of AD patients have late onset "sporadic" AD. Only 10% become symptomatic before age 65. Two percent of AD is familial and is caused by autosomal dominant genetic mutations. AD is caused by the aberrant folding and aggregation of two proteins: β-amyloid and tau. The disease is also characterized by synaptic dysfunction and neuronal loss. Protein metabolic disturbances lead to a variety of secondary cell injury and death mechanisms including oxidation, inflammation, excitotoxicity, and apoptosis. Emerging therapies are largely directed at amyloid beta protein production or accumulation or the cascade of secondary consequences. The current U.S. FDA-approved therapy includes the anticholinesterase inhibitors and the N-methyl-D-aspartate blocker memantine. New drug trials with disease-modifying agents are currently on their way. [ABSTRACT FROM AUTHOR]

Considerable advances have been made in the past years in developing novel experimental models of neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Fronto-Temporal Dementias (FTD), Amyotrophic Lateral Sclerosis (ALS), and trinucleotide repeat disorders (TNRDs). The main postulate of several of the genetically modified murine models of neurodegenerative disorders is that a single molecular alteration might trigger a cascade of events that eventually will result in the full spectrum of the clinicopathological alterations observed in human disease. Therefore, overexpression of mutant proteins in transgenic (tg) mice might mimic some aspects associated with the gain of a toxic function, while targeted deletion of selected genes might mimic aspects associated with loss of a trophic or protective function. To date, several genes have been identified to be associated with familial forms of AD, PD, FTD, ALS, and TNRDs. Single or combined tg and knockout models targeting most of these genes have been developed that recapitulate one or several aspects of each disorder. In these models, abnormal accumulation and misfolding (toxic conversion) of endogenous proteins is being extensively explored as a key pathogenic event leading to neurodegeneration. Thus, the main focus of this chapter is to provide a perspective as to the efforts in developing genetically engineered models of the most common neurodegenerative disorders. Further development and investigation of animal models of these diseases holds the promise of better understanding their pathogenesis and discovering and testing new treatments. [ABSTRACT FROM AUTHOR]

Defining specific mutations in familial human neurodegenerative diseases has allowed researchers to make animal models of the diseases through directed genetic approaches. These studies help address the molecular mechanisms of disease, and provide the foundation toward therapeutics. Modeling human neurodegenerative diseases in invertebrates has revolutionized the field in such a way that both reverse and forward genetic approaches are leading to the discovery of new players in neurodegeneration. This review focuses on fruit fly and nematode models of human neurodegenerative diseases, with emphasis on how these models have provided new insights into aspects of human disease. [ABSTRACT FROM AUTHOR]

Amyloid fibril formation is a phenomenon common to many proteins and peptides associated with numerous conformational diseases. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. This chapter describes a method to visualize amyloid fibril growth in real time at the single fibril level. This approach uses total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloidspecifi c fluorescence dye. The method enables an exact analysis of the rate of growth of individual fibrils. One of the advantages of TIRFM is that only amyloid fibrils lying in parallel with the slide glass surface were observed, so that one can obtain the exact length of fibrils. This method is of particular importance for the analysis of rapid fibrillation kinetics, providing unique information crucial for the elucidation of the molecular mechanisms of amyloid fibril formation. [ABSTRACT FROM AUTHOR]

The ultimate objective of the amyloid fibril studies is to elucidate the physicochemical aspects and molecular mechanisms of pathological self-assembly of biological macromolecules. In situ atomic force microscopy (AFM) is a useful tool in studying the aggregation of peptides associated with various conformational diseases under a wide variety of conditions. The unique capability of in situ AFM is the direct visualization of the behavior of biological macromolecules at solid-liquid interfaces, under nearly physiological conditions. AFM can provide information that is not easily accessible by other methods due to the unique ability to follow in time three-dimensional nanoscale surface maps. It is of particular importance for the analysis of the roles of surfaces (including supported lipid bilayers and cells) in the processes leading to pathological peptide assembly and the structural consequences of the addition of modulating factors on such aggregation. Used in conjunction with other techniques, AFM has become an invaluable tool, providing useful information about disease-related protein aggregation. [ABSTRACT FROM AUTHOR]

Amyloid fibrils are insoluble aggregates that result from the self-assembly of partially unfolded proteins. Regardless of the native structure of the precursor proteins, the predominant secondary structure in the fibrillar form is β-sheet. Proteins that form amyloid in vivo are associated with numerous diseases, including Alzheimer's, Parkinson's, and prion diseases. The three-dimensional structures of amyloid fibrils may provide valuable information on the misfolded protein conformations and on the pathways that lead to disease. The purpose of this chapter is to introduce electron microscopy as a tool for the structure determination of amyloid fibrils. [ABSTRACT FROM AUTHOR]

Reporters of amyloid fibril structure have contributed greatly to our understanding of the biochemical processes of fibril formation. Congo Red and Thioflavine T have been used to discover agents that disrupt fibril formation. Now labeled tight binding ligands are being assessed for their utility as imaging agents to diagnose amyloid deposition and to monitor the efficacy of therapeutic regimens. Antibodies selective for the fibrils or amyloidogenic forms of the proteins originally defined by microscopy and by small molecule probes are presently being used to investigate the involvement of these species in the pathology of disease. Some of these immunological tools may also have therapeutic utility in depleting toxic forms of the proteins. Further development of these probes will improve diagnosis and expedite the development of therapeutic strategies for the increasing number of recognized diseases of protein misfolding. [ABSTRACT FROM AUTHOR]

The localization of amyloid fibril components and the cells related to the formation and resorption of the fibrils are still controversial. In this study we undertook a time-kinetic study to analyze the process of amyloid fibril deposition in the spleen of AA amyloidosis animal model immunohistochemically and ultrastructurally. Murine amyloid A (AA) amyloidosis was induced by the emulsion injection composed of Freund's complete adjuvant and Mycobacterium butyricum. Serum amyloid A (SAA) level was the highest at 3 days after the induction and gradually decreased. The amyloid deposition was first detected in extracellular spaces in the marginal zone of the spleen at 7 days after induction. The F4/80 positive red pulp macrophages increased in number after the induction and accumulated near the amyloid deposition areas. Amyloid P component (APC) and chondroitin sulfate proteoglycan (CSPG), which are composed of amyloid fibril, were detected in the cytoplasm of F4/80 positive red pulp macrophages and ER-TR9-positive marginal zone macrophages, respectively, then localized in the amyloid deposition areas. APC was also localized in CSPG-positive and F4/80-negative cells, which might be fibroblasts at 3 days. Ultrastructural examination indicated that macrophages in the marginal zone contained lysosome-derived fibrillar structures of amyloid, and that fibroblasts extended amyloid fibrils into the extracellular area in the marginal zone. These results suggested the close association of APC-positive/ER-TR9-positive macrophages and APC-positive/CSPG-positive fibroblasts with the formation of amyloid fibrils and F4/80-positive macrophages with the resorption of the fibrils. [ABSTRACT FROM AUTHOR]

Congo red (CR) is the most popular dye used as a probe for diagnosing amyloidosis, a very heterogeneous group of diseases with more than 23 chemically different amyloid syndromes of men and animals, leading to more than 400 different individual diseases. Congo red binding increases the natural anisotropy of amyloid, indicating that the elongated and planar CR molecules are aligned parallel to the axis of the amyloid α fibrila and to each other, thereby revealing a structure of amyloid. This structure was established to represent fibrils of similar dimensions, although the amyloid fibrils can be composed of many unrelated proteins. This CR-induced (positive) anisotropy displaying a green color is the hallmark of all amyloids, and is therefore used in the diagnosis of amyloidosis. The specificity of this criterion, however, is based on very stringent conditions of staining and evaluation. This review will focus on the understanding of the CR staining procedure, its mechanism and, in particular, on its recent practical improvements by increasing the sensitivity of the CR procedure, so that minute and the earliest amyloid deposits in the course of amyloidosis can now be reliably detected in patients. This enables a very early diagnosis in the course of the disease before irreversible organ damage might have occurred, and widens the options for a successful therapy. In addition, the central role of the CR diagnostic procedure and evasion of common pitfalls in arriving at a pathogenetically exact classification must only be based on the chemical nature of the amyloid deposits and not on the soluble precursors of the many different amyloidoses will be highlighted. The proposed bench-tobedside algorithm will enable the physician to arrive at the exact diagnosis for therapeutic considerations. Finally, some possible future applications of CR and analogues will be presented. [ABSTRACT FROM AUTHOR]

A plethora of clinical syndromes are characterized by the deposition of amorphous, Congo red staining material known as "amyloid." These protein folding diseases include Alzheimer's, Parkinson's, type II diabetes mellitus, rheumatoid arthritis, and "mad cow" disease. Amyloid-forming peptides readily adapt beta-sheet structure and can spontaneously aggregate into extended fibrils despite having no primary sequence homology. All amyloid peptides appear to interact strongly with lipid membranes, assemble into oligomers, and form ion-permeable channels. These channels are large, heterogeneous, nonselective, and irreversible. They are inhibited by Congo red and blocked by Zn+2. The leakage pathway induced by these channels could be responsible for the cellular pathology of amyloidoses, including membrane depolarization, mitochondrial dysfunction, inhibition of long-term potential (LTP), and cytotoxicity. We suggest that channel formation underlies amyloid disease. [ABSTRACT FROM AUTHOR]

The accumulation of misfolded protein into insoluble inclusions is a pathological hallmark of many diseases. How these inclusions form and their role in the degenerative process is still unknown. Recently, a cellular response to the accumulation of misfolded protein was described, and the resulting structures were termed Aggresomes. Aggresomes occur in cells due to impairment of intracellular degradation pathways, and are insoluble inclusions associated with a rearranged intermediate filament network. Aggresomes form by the microtubule and dynein-dynactin-dependent delivery of small microaggregates of protein to a central cellular location, in most cases the centrosome. In this chapter, the characteristics of aggresomes are described, followed by a discussion of the relationship of aggresomes to the general class of ubiquitin-intermediate filament diseases (most notably characterized by Lewy Bodies in Parkinson's Disease, intranuclear inclusions in Huntington's Disease, and Bunina Bodies in amyotrophic lateral sclerosis), and conclude with potential mechanisms for aggresome formation. Aggresomes have the potential to provide a mechanistic clue to the pathogenesis of ubiquitin-intermediate filament disorders. [ABSTRACT FROM AUTHOR]

Overwhelming experimental evidence supports the hypothesis that molecular chaperones are critical modulators of protein aggregation and toxicity in a number of protein misfolding diseases. However, the mechanism by which chaperone activity facilitates neuroprotection remains poorly understood. Early intermediates in the assembly process of Aβ aggregates have been found to be potent neurotoxins in vivo, and it is likely that prefibrillar intermediates of other disease proteins may have similar pathogenic effects. Accordingly, a key step in the pathogenesis of the various proteinopathies may stem from the aberrant interactions of altered protein conformations or prefibrillar intermediates with key cellular proteins, effectively sequestering their activity and triggering a cascade of events that culminates in neuronal dysfunction prior to the appearance of inclusions. The vast majority of animal studies have shown that chaperones facilitate neuroprotection in the absence of a visible effect on inclusion formation, suggesting that protective interactions may occur at the level of prefibrillar aggregation intermediates, or by preventing conformational changes that precede the formation of aggregation intermedites. It will be important to develop techniques that enable in vivo detection of early aggregation intermediates for the various protein misfolding diseases and determine how interaction of these intermediates with other cellular proteins, such as the molecular chaperones, alters pathogenesis. Ultimately, it is necessary to understand how the various components of the protein quality proteome work together to regulate the toxicity of misfolded proteins. Effective therapies will likely require the simultaneous modulation of numerous components of the cellular quality control apparatus, and the molecular chaperones will play a key role in these types of approaches. Because the molecular chaperones provide a first line of defense against misfolded proteins, and are likely to function at the earliest stages of disease pathogenesis, they are a particularly exciting prospect for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Protein destabilization by mutations or external stresses may lead to misfolding and aggregation in the cell. Often, damage is not limited to a simple loss of function, but the hydrophobic exposure of aggregate surfaces may impair membrane functions and promote the aggregation of other proteins. Such a "proteinacious infectious" behavior is not limited to prion diseases. It is associated to most protein-misfolding neurodegenerative diseases and to aging in general. With the molecular chaperones and proteases, cells have evolved powerful tools that can specifically recognize and act upon misfolded and aggregated proteins. Whereas some chaperones passively prevent aggregate formation and propagation, others actively unfold and solubilize stable aggregates. In particular, ATPase chaperones and proteases serve as an intracellular defense network that can specifically identify and actively remove by refolding or degradation potentially infectious cytotoxic aggregates. Here we discuss two types of molecular mechanisms by which ATPase chaperones may actively solubilize stable aggregates: (1) unfolding by power strokes, using the Hsp100 ring chaperones, and (2) unfolding by random movements of individual Hsp70 molecules. In bacteria, fungi, and plants, the two mechanisms are key for reducing protein damages from abiotic stresses. In animals devoid of Hsp100, Hsp70 appears as the core element of the chaperone network, preventing the formation and actively removing disease-causing protein aggregates. [ABSTRACT FROM AUTHOR]

Formation of amyloid fibrils from an amyloidogenic precursor peptide is a stochastic process. Initially, conditions do not favor aggregation, and this period corresponds to the lag phase that precedes the possible formation of fibrils. This lag phase can be overcome by the amyloidogenic peptide reaching a critically high concentration, or via binding with apolipoproteins or other chaperone proteins. Apolipoproteins interact with numerous amyloidogenic peptides in diverse systemic and organ limited amyloidoses because of their natural tendency to bind hydrophobic domains present on these peptides. Apolipoproteins appear to play an essential role in the kinetics of amyloid deposition. Apoliprotein E (apo E), which has been found to be involved in the majority of amylodoses, helps to overcome the initially unfavorable kinetic barrier of amyloid fibril formation, and typically shows isoform-specific effects that influence the epidemiology, clinical course, and pathological aspects of disease. The modulatory effect of apolipoproteins may not be evident if either the amyloid-prone protein is present in great excess or carries a mutation rendering it extremely amyloidogenic. Under these conditions amyloid deposition can occur without the help of chaperones. The exact binding site of amyloidogenic peptides on apo E and other apolipoproteins is unclear, and it remains unknown if it is a linear or a conformational epitope. Mapping this site could provide a means to clarify the mechanism of interaction between apolipoproteins and their peptide ligands, as well as allowing the generation of peptidomimetic compounds that block this interaction in an effort to develop successful treatment approaches. It appears that apo E has one universal site interacting with various amyloidogenic peptides regardless of their sequence. Targeting the pathological chaperones of amyloidoses such as apo E appears to be an attractive approach from a pharmacokinetic point of view, because the concentration of these molecules in amyloid deposits is usually 100 to 200 times lower than the concentration of the actual fibril-forming peptide (Ma et al., 1994; Wisniewski et al., 1994a). In addition, blocking this interaction between apo E and amyloidogenic proteins is unlikely to be associated with any significant toxicity, making this a significant therapeutic target. [ABSTRACT FROM AUTHOR]

Despite divergent opinions on whether protein inclusions represent a protective or harmful mechanism in the progression of diseases such as Alzheimer's disease, Parkinson's disease, and prion diseases, oxidative and nitrative modifications may play a critical role in development of the diseases. Oxidative and nitrative modifications may occur early during the disease initiation, or later, compromising cellular functions once the inclusions have become sufficiently large at later stages of disease progression. These assertions are supported by exciting published but preliminary evidence, which requires extensive validation by experiments employing simple in vitro systems, cellular and animal models. Experiments in progress should inform us of the significance of oxidative and nitrative chemistries in the pathological mechanisms of diseases characterized by protein inclusions. [ABSTRACT FROM AUTHOR]

Increasing evidence exists to support a potentially crucial role for proteoglycans, in particular heparan sulfate proteoglycans, in the pathophysiology of many protein conformational disorder diseases. This chapter will focus on emerging evidence that supports a role for proteoglycans in the regulation of protein conformation in amyloid diseases, with the result being that proteoglycans are attractive therapeutic targets for these diseases. Amyloid diseases in both the nervous system and nonneural tissues share a common property with proteoglycans, especially heparan sulfate proteoglycans, being associated with amyloid fibrils coincident with the time amyloid fibrils are formed in the disease process. The binding of heparan sulfate proteoglycans to the specific amyloidassociated protein has been shown in many cases to lead to conformational changes in the amyloidassociated protein, with an introduction of β-sheet structure to the amyloid protein. Heparan sulfate proteoglycans have also been demonstrated to accelerate the formation of amyloid oligomers, protofibrils, and fibrils, as well as impart resistance to proteolytic degradation of the amyloid fibrils. Heparan sulfate proteoglycans therefore appear to play a critical role in the progression of amyloid diseases, and an ability to prevent heparan sulfate binding to amyloid-associated proteins may allow both an inhibition of the formation of new amyloid, as well as promote the clearance of existing amyloid. [ABSTRACT FROM AUTHOR]

Multiple diseases, as diverse as diabetes and mad cow disease, exhibit accumulations of abnormal protein fibrils. Generically referred to as "amyloid," these self-assembling fibrils typically have been considered the pathogenic molecules that cause cellular degeneration (toxins, not just tombstones). A prominent example is the "amyloid cascade hypothesis" proposed for Alzheimer's disease (Hardy and Higgins, 1992). Fibrils, however, are not the only toxins generated by protein self-association, probably in some cases not even the most relevant ones. We now know of toxic subfibrillar species—soluble oligomers and protofibrils. The emerging hypothesis considered here is that these novel subfibrillar assemblies, the hidden toxins, constitute significant pathogenic molecules in diseases of fibrillogenic proteins. Clues leading to this hypothesis have come in many instances from studies of Aβ (Klein et al., 2001), the fibrillogenic peptide responsible for amyloid plaques in Alzheimer's disease (AD). Alzheimer's disease is the most common form of dementia in the elderly, affecting 10% of individuals older than 65 (Hebert et al., 2003) and more than 25 million individuals world-wide. This chapter examines the investigation of Aβ's role in AD essentially as a case study. Its objectives are to (1) review the link between Alzheimer's dementia and fibrillogenic proteins and show that pathogenesis truly involves Aβ; (2) show how key problems in the amyloid cascade hypothesis disappear with the discovery of subfibrillar Aβ assemblies; (3) discuss cellular mechanisms of the new toxins that explain why AD is a disease of memory loss; (4) consider data that clinically substantiate a new, oligomerinitiated amyloid cascade hypothesis; (5) assess whether the impact of subfibrillar toxins can provide a broad mechanism applicable to multiple fibrillogenic proteins; (6) evaluate emerging implications for therapeutics and diagnostics. At present, no cause for AD has been established; there are no effective therapeutics, and no clinical diagnostics exist. This situation, however, is rapidly changing. New insights into disease mechanisms and remarkable new therapeutic antibody strategies give us cause for optimism. [ABSTRACT FROM AUTHOR]

The ability of proteins to fold to their functional states is an astonishing example of the power of biological evolution at the molecular level. Despite the large number of different native protein folds, the process of folding can be described in terms of a universal mechanism that appears to be based on the generation of the correct overall topology through interactions involving a relatively small number of residues. Protein misfolding is an intrinsic aspect of normal folding within the complex cellular environment, and its effects are minimized in living systems by the action of a range of protective mechanisms including molecular chaperones and quality control systems. Unfolded and misfolded proteins have a tendency to aggregate to form a variety of species including the highly organized and kinetically stable amyloid fibrils. The latter species represent a generic form of structure resulting from the inherent polymer properties of polypeptide chains, and their formation is associated with a wide range of debilitating human diseases. Amyloid fibrils and their precursors appear to have similar adverse effects on cellular function regardless of the sequence of the component peptide or protein. Our increasing knowledge of the interplay between different forms of protein structure and their generic characteristics provides a platform for rational therapeutic intervention designed to prevent or treat this whole family of diseases. [ABSTRACT FROM AUTHOR]

A full understanding of the mechanism by which proteins and peptides convert from their soluble states into amyloid aggregates requires a detailed elucidation of the sequence and structural determinants that govern the processes of amyloid formation. The experimental results collected in the past few years converge on the idea that hydrophobicity, propensity to form a secondary structure and charge are key determinants of aggregation. The effect of mutations on aggregation and the reasons why particular regions of the sequence are more effective than others in promoting aggregation of unstructured polypeptide chains can be explained on the basis of these physicochemical factors. At present, it is possible to edit algorithms to calculate the relative effects of mutations on aggregation, the absolute aggregation rate of a natively unfolded protein, and to identify regions of the sequence that play key roles in promoting the aggregation process of the whole protein. In addition, because amyloid formation seems to be a shared property of natural proteins, protein sequences appear to have evolved to reduce their propensities to aggregate. One of the strategies by which proteins have achieved this end has been to modulate their aggregation potential by playing with these factors. [ABSTRACT FROM AUTHOR]

Recent reports give strong support to the idea that amyloid fibril formation and the subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. In this review recent findings are surveyed to illustrate that protein fibrillogenesis requires a partially folded conformation. This amyloidogenic conformation is relatively unfolded, and shares many structural properties with the premolten globule state, a partially folded intermediate frequently observed in the early stages of protein folding and under some equilibrium conditions. The inherent flexibility of such an intermediate is essential in allowing the conformational rearrangements necessary to form the core cross-β; structure of the amyloid fibril. [ABSTRACT FROM AUTHOR]