Amyloidosis and the .beta.-Amyloid Peptide
Amyloidosis generally refers to a physiological condition which involves deposition of insoluble polypeptides, termed amyloid polypeptides or amyloid proteins. There are a wide range of amyloid proteins found in various tissues throughout a subject, and a number of pathological conditions associated with various amyloidoses. For example, multiple myeloma can result in amyloidosis with the immunoglobulin proteins. Idiopathic familial Mediterranean fever also involves systemic amyloidosis. Perhaps the best known disease associated with amyloidosis is Alzheimer's disease.
Alzheimer's disease (AD) affects more than 30% of humans over 80 years of age, and as such, represents one of the most important health problems of developed countries (Evans et al., 1989, JAMA 262:2551-56; Katzman and Saitoh, 1991, FASEB J. 280:278-286). The etiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated with deposits of amyloid plaques, cerebrovascular amyloid and neurofibrillary tangles in the brain and cerebrovasculature. The number of plaques in AD patients' brains are typically 5- to-10 fold greater than in age-matched healthy controls. Increased levels of plaques may result from increased rate of synthesis of the components of the plaques, decreased rate of degradation, or some combination of the two.
The primary protein component of plaques is the 42 amino acid (4.2 kDa) beta-Amyloid Peptide (.beta.AP), which derives from a family of larger Amyloid Peptide Precursor (APP) proteins (Glenner and Wong, 1984, Biochem. Biophys. Res. Commun. 120:885-890; Glenner and Wong, 1984, Biochem. Biophys. Res. Commun. 122:1131-35; Goidgaber et al., 1987, Science 235:8778-8780; Kang et al., 1987, Nature 325:733-736; Robakis et al., 1987, Proc. Natl. Acad. Sci. USA 84:4190-4194; Tanzi et al., 1987, Science 235:880-884). The process of amyloidosis is poorly understood, but requires at least .beta.AP. Recent evidence shows that .beta.AP may be found in extracellular spaces like cerebrospinal fluid (CSF) of the brain and conditioned media of many cell types. Since increased amounts of amyloid deposits are present in AD brains one simple hypothesis is that increased .beta.AP production leads to increased amyloidosis. Messenger RNAs encoding the APP precursors of .beta.AP increase about 2-fold in AD brains, which has suggested to some a possible 2-fold increase in rates of translation, which may explain increased amyloid plaque formation (e.g., Jacobsen et al., 1991, Neurobiol. Aging 12:585-592, and references cited therein; Palmert et al., 1989, Prog. Clin. Biol. Res. 317:971-984; Tanaka et al., 1990, Rinsho Byori 38:489-493; Tanaka et al., 1989, Biochem. Biophys. Res. Commun. 165:1406-1414). An example of an increased efficiency of .beta.AP production that correlates with increased plaque levels is found in a rare genetically linked familial form of Alzheimer's disease (Cai et al., 1992, Science 259:514-516; Citron et al., 1992, Nature 360:672-674; Mullan et al., 1992, Nature Genet. 1:345-347), known as a Swedish disease involving a double lysine-methionine (KM) to asparagine-leucine (NL) mutation in APP near the amino-terninus of .beta.AP. This mutation increases the release of extracellular .beta.AP in cultured cells. However, while this observation may partly explain amyloidosis in the Swedish disease (and Down's Syndrome), .beta.AP peptide levels in CSF of AD and healthy patients are the same (Oosawa et al., 1993, Soc. Neurosci. Abst. 19:1038; Shoji et al., 1992, Science 258:126129). Thus, although healthy subjects appear to possess similar quantities of .beta.AP as AD patients, they nevertheless fail to accumulate the high number and amount of amyloid plaques seen in their AD counterparts.
Post-translational events may contribute to amyloidosis. Beyond increased rates of translation, physiological events such as greater efficiency of .beta.AP production from its precursor, aggregation into fibrillar structures, and resistance to proteolysis may unbalance degradative processes, resulting in plaque formation.
Aggregation of the components of amyloid is a critical step in the development of amyloidosis. Once formed, fibrillar aggregates of .beta.AP are extremely stable and not easily degraded. Amyloid plaques may be purified by their resistance to solubilization in boiling SDS and digestion with a variety of proteases. Additional treatment with 80% formic acid or 6M guanidine thiocyanate eventually solubilizes a portion of the plaque material. The solubilized protein is primarily the 42 amino acid .beta.AP. Yet even after these harsh denaturation treatments, dimers, tetramers and large molecular weight aggregates containing immunoreactive .beta.AP are found. This resistance to solubilization into soluble or monomeric components suggests extensive protein modifications.
Further experiments have shown that primary neuronal cultures treated with full length .beta.AP 1-42 in soluble form remain viable. Thus, soluble .beta.AP 1-42 shows no toxicity. In contrast, cultures treated with insoluble aggregates of .beta.AP 1-42 show a toxic response (Pike et al., 1991, Eur. J. Pharm. 207:367-368; Pike et al., 1993, J. Neurosci. 13:1676-87). This experiment suggests that the toxicity of .beta.AP is related to its state of aggregation. Thus, an understanding of the mechanism forming fibrils and/or insoluble aggregates from soluble .beta.AP may be critical to preventing toxicity and resulting neurodegenerative disease.
In the absence of increased soluble .beta.AP in most cases of AD, the question remains how amyloid accumulates to a greater degree at different rates. Synthetic .beta.APs corresponding to the first 28, 40, or 42 amino acids of .beta.AP (i.e., .beta.AP 1-28, .beta.AP 1-40 and .beta.AP 1-42, respectively) display concentration-dependent aggregation kinetics in in vitro incubations. Fibrillar aggregates form in vitro and these appear similar to brain .beta.-amyloid fibrils at the morphological level using electron microscopy and at the light microscopy and spectroscopic levels using Congo Red and Thioflavin stains.
The more rapid kinetics of aggregation observed at .mu.M concentrations of soluble .beta.AP in vitro are only of limited relevance for insight into the mechanism of fibril formation in vivo. At lower .beta.AP concentrations, for instance in the physiological range of about 5 nM, there is a considerable lag period before measurable aggregate is formed in vitro. This observation suggests that the rate limiting step in aggregation could be formation of a "nucleus" or "seed" upon which additional .beta.AP can rapidly accumulate.