Proteolytic degradation of the amyloid beta-protein: the forgotten side of Alzheimer's disease

Proteases have long played a central role in the molecular pathogenesis of Alzheimer's disease (AD), yet proteases that degrade the amyloid beta-protein (Abeta) itself were largely ignored until only quite recently. Today, we know that Abeta-degrading proteases are critical regulators of brain Abeta levels in vivo, with evidence accumulating that their dysfunction may play a role in the etiology of AD. This review explores the historical factors that obscured this important aspect of amyloidogenesis, and discusses the many fresh insights it offers into the causes of and potential treatments for AD.     

Computer simulations of Alzheimer's amyloid beta-protein folding and assembly

Pathological folding and aggregation of the amyloid beta-protein (Abeta) are widely perceived as central to understanding Alzheimer's disease (AD) at the molecular level. Experimental approaches to study Abeta self-assembly are limited, because most relevant aggregates are quasi-stable and inhomogeneous. In contrast, simulations can provide significant insights into the problem, including specific sites in the molecule that would be attractive for drug targeting and details of the assembly pathways leading to the production of toxic assemblies. Here we review computer simulation approaches to understanding the structural biology of Abeta. We discuss the ways in which these simulations help guide experimental work, and in turn, how experimental results guide the development of theoretical and simulation approaches that may be of general utility in understanding pathologic protein folding and assembly.     

Immunohistochemical localization of amyloid beta-protein with amino-terminal aspartate in the cerebral cortex of patients with Alzheimer's disease

We investigated immunohistochemically the localization of amyloid beta-protein (Abeta) with amino-terminal aspartate (N1[D]) in brains of patients with Alzheimer's disease, diffuse Lewy body disease and Down's syndrome. A monoclonal antibody, 4G8, which recognizes the middle portion of Abeta, was used as a reference antibody to label the total Abeta deposits. Double staining with anti-Abeta(N1[D]) and 4G8 revealed that Abeta deposits in the subiculum and the neocortical deep layers often lacked N1[D] immunoreactivity, indicating N-terminal truncation of Abeta in these deposits. Abeta deposits in the neocortical superficial layers and the presubicular parvopyramidal layer always contained Abeta with N1[D]. Such regional as well as laminar differences in the distribution of Abeta beginning at N1[D] suggest that some local factors influence N-terminal processing of Abeta deposited in the brain.     

Nicotine lowers the secretion of the Alzheimer's amyloid beta-protein precursor that contains amyloid beta-peptide in rat

Reports of an inverse relationship between nicotine intake, due to cigarette smoking, and the incidence of Alzheimer's disease (AD) prompted us to investigate the effects of nicotine on amyloid beta-protein precursor (AbetaPP) processing in rat. Over-production and/or altered metabolism of AbetaPP, resulting in increased amyloid beta-peptide (Abeta), appear pivotal in the pathogenesis of AD. Abeta is generated proteolytically from betaPP by a group of secretases. AbetaPP cleavage by gamma-secretase results in the secretion of a truncated soluble betaPP (sAPPgamma) that contains intact Abeta. Nicotine, 1 and 8 mg/kg/day, doses commensurate with cigarette smoking and a higher but well tolerated dose, respectively, was administered over 14 days and Western blot analysis was performed on sAPP fragments. Both doses significantly reduced sAPPgamma. These actions were blocked by nicotinic receptor antagonism. Whereas nicotinic antagonists alone had no effect on either total sAPP or sAPPgammalevels in CSF, muscarinic antagonism significantly elevated them; suggesting that muscarinic rather than nicotinic receptor silence alters processing of AbetaPP to favor a potentially amyloidogenic route. Combined nicotine and muscarinic antagonism attenuated the action of the latter to elevate sAPPgamma, indicating that nicotine modifies AbetaPP processing away from potentially amyloidogenic products. These results suggest that within the brain, levels of total sAPP, sAPPgamma and, accordingly, Abeta are subject to cholinergic manipulation, offering therapeutic potential at the level of AbetaPP processing to decrease Abetadeposition.     

Cerebellar amyloid plaques in Alzheimer's disease

In a consecutive series of 30 brains of demented patients (presenile, senile and familial types) with the histological hallmarks of Alzheimer's disease, cerebellar amyloid plaques and cerebellar amyloid angiopathy were observed in 80% of the cases. These cerebellar amyloid plaques were sometimes centered on a small amyloidotic blood vessel. They were immunostained with A4 antiserum, but they were not surrounded by a crown of swollen neurites as demonstrated with silver impregnation and Tau antiserum. They were not immunostained with SAF antiserum which decorated the cerebellar Kuru-like plaques observed in subacute transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome. The absence of neuritic changes around the numerous cerebellar amyloid plaques frequently observed in cases of Alzheimer's disease is an interesting feature and will perhaps explain the mechanism of cytoskeleton changes occurring in the neurons of the cerebral cortex.     

Clinical amyloid imaging in Alzheimer's disease

BACKGROUND: The hypothesis that amyloid deposition is the leading cause of Alzheimer's disease (AD) is supported by findings in transgenic animal models and forms the basis of clinical trials of anti-amyloid agents. According to this theory, amyloid deposition causes severe damage to neurons many years before onset of dementia via a cascade of several downstream effects. This hypothesis has, however, not yet been directly tested in human beings because of the very limited possibility of diagnosing amyloid deposition in vivo, which until recently required either brain biopsy or PET imaging with an on-site cyclotron and radiochemistry laboratory. Moreover, a clinical diagnosis of AD requires that patients have dementia, at which stage any effective treatment aimed at reducing amyloid deposition will probably be too late. RECENT DEVELOPMENTS: The amyloid imaging tracers flutemetamol, florbetapir, and florbetaben labelled with (18)F have been developed for PET; they can be produced commercially at central cyclotron sites and subsequently delivered to clinical PET scanning facilities. These tracers are currently undergoing formal clinical trials to establish whether they can be used to accurately image fibrillary amyloid and to distinguish patients with AD from normal controls and those with other diseases that cause dementia. They might also be used as biomarkers to predict development of AD before onset of dementia and to assess the effect of anti-amyloid therapy. Negative amyloid scans indicate absence of AD with a high level of accuracy, but healthy elderly volunteers might have positive amyloid scans, so their predictive value in isolation is less clear. Close association of in-vivo amyloid imaging results with post-mortem histopathological findings was shown with florbetapir in a phase 3 study. WHERE NEXT?: Therapeutic studies of anti-amyloid agents that include amyloid tracers as biomarkers are expected to be useful for drug development and to clarify the relation between amyloid removal and clinical effects. Once the (18)F tracers become available for diagnostic use, large-scale longitudinal studies will be needed to clarify their prognostic and diagnostic power in relation to age, risk factors, and AD subtypes. Ultimately, these tracers will hopefully clarify the pathophysiological role of amyloid in AD and contribute to development of new treatments.     

Genetic aspects of Alzheimer's disease, Pick's disease, and other dementias

Although genetic testing is available for some degenerative diseases, in most types of dementia, both genetic and environmental factors are involved. Overall, dementing diseases can be either sporadic or inherited, and in general, the earlier the onset, the more likely a disease is to be inherited. Before genetic testing is performed, the ethical issues, such as the effect the tests might have on asymptomatic children, should be considered. The ethical use of DNA samples in research is another genetic testing issue to be considered.     

PET imaging of amyloid in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia and is characterised by progressive impairment in cognitive function and behaviour. The pathological features of AD include neuritic plaques composed of amyloid-beta peptide (Abeta) fibrils, neurofibrillary tangles of hyperphosphorylated tau, and neurotransmitter deficits. Increases in the concentration of Abeta in the course of the disease with subtle effects on synaptic efficacy will lead to gradual increase in the load of amyloid plaques and progression in cognitive impairment. Direct imaging of amyloid load in patients with AD in vivo would be very useful for the early diagnosis of AD and the development and assessment of new treatment strategies. Three different strategies are being used to develop compounds suitable for in vivo imaging of amyloid deposits in human brains. Monoclonal antibodies against Abeta and peptide fragments have had limited uptake by the brain when tested in patients with AD. When putrescine-gadolinium-Abeta has been injected into transgenic mice overexpressing amyloid, labelling has been observed with MRI. The small molecular approach for amyloid imaging has so far been most successful. The binding of different derivatives of Congo red and thioflavin has been studied in human autopsy brain tissue and in transgenic mice. Two compounds, fluorine-18-labelled-FDDNP and carbon-11-labelled-PIB, both show more binding in the brains of patients with AD than in those of healthy people. Additional compounds will probably be developed that are suitable not only for PET but also for single photon emission CT (SPECT).     

Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type share a decrease in cerebrospinal fluid levels of amyloid beta-protein precursor.

The amyloid beta-protein is a 39-42 amino acid peptide that is deposited in senile plaques and in cerebral vessel walls in individuals with Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and, to a much lesser extent, normal aging. It is derived from abnormal proteolytic processing of its parent protein, the amyloid beta-protein precursor. Here we show that individuals with the HCHWA-D mutation and clinically manifesting the disease have markedly decreased cerebrospinal fluid levels of soluble amyloid beta-protein precursor (0.7 +/- 0.4 micrograms/ml) compared with age-matched normal subjects (3.0 +/- 0.2 micrograms/ml) as determined by quantitative immunoblotting and enzyme-linked immunosorbent assays. Similarly, age-matched patients diagnosed with probable Alzheimer's disease also have decreased cerebrospinal fluid levels of soluble amyloid beta-protein precursor (1.0 +/- 0.3 micrograms/ml). These parallel findings suggest a common biochemical marker for these two diseases and further establish the pathogenic relatedness of HCHWA-D and Alzheimer's disease.     

White matter amyloid in Alzheimer's disease brain

Amyloid -protein (A) deposits in the white matter were investigated by the double immunohistochemical staining for A and neuritic, glial or vascular components. Reactive astroglia and neurite abnormality were absent around A deposits in the white matter (w-A) even those with a core. The association of w-A with blood vessels was not consistent. Aggregates of activated microglia were found to be the sole but a consistent accompaniment of A deposits even in the absence of other components such as neuron, synapse, neurite abnormality and reactive astroglia, as observed in the white matter. This suggests that the aggregates of activated microglia most likely represent one of the factors promoting the process of A deposition.     

Genotoxicity in Alzheimer's disease: role of amyloid

Alzheimer's disease (AD) is a complex neurodegenerative disorder pathologically identified by the presence of extracellular senile plaques (SP) with a proteinaceous core composed of aggregates of the amyloid peptide (Abeta) and intracellular aggregates of the microtubule-associated protein tau (tau) as neurofibrillary tangles (NFTs). These hallmarks consist of abnormally folded proteinaceous components that are believed to be neurotoxic in AD. The mechanisms of toxicity remain unclear although oxidative stress and inflammation are implicated as mediators of the toxicity and these lesions, in turn, are known to damage cellular components including proteins, lipids in the membrane and DNA. However effects on genotoxicity and its role in AD are less clear. The present review discusses various influences, in particular of amyloid, on the genetic material and their possible role in the neurodegeneration in AD. Further, the amalgamation of genomics and proteomics in understanding AD and therapeutic development is suggested.     

Ultrastructural heterogeneity in cerebral amyloid of Alzheimer's disease

Summary Cerebral amyloid deposits from five patients with presenile or senile cerebral disease of the Alzheimer type were stained with uranyl acetate and lead citrate or with periodic acid-thiocarbohydrazidesilver proteinate, and examined with traditional highresolution electron microscopy and with a goniometer tilting stage. In addition to a carbohydrate-rich matrix, we also consistently found local cell-derived vesicles within plaque and dyshoric amyloid. The most likely source for these vesicles appeared to be degenerate neurites. Amyloid fibrils were intimately associated with plasmalemmata, particularly those of degenerate neurites, which supported a neuronal origin for the amyloid fibril of Alzheimer's disease.     

Neuropsychological aspects of Alzheimer's disease

This analysis is centered on the study of cognitive disorders in Alzheimer's Disease (AD), mainly for major neuropsychological functions. We insist on the heterogeneity of the clinical picture especially in the early stages of the illness, when deficits of episodic memory and executive functions are prevalent. We consider that studying early stages of the illness is necessary to delineate the diagnostic signs, to validate the new therapeutic experiments, to predict stages of decline.     

Genetic studies in Alzheimer's disease

Alzheimer's disease (AD), the most common cause of dementia in aged populations, is believed to be caused by both environmental factors and genetic variations. Extensive linkage and association studies have established that a broad range of loci are associated with AD, including both causative and susceptibility (risk factor) genes. So far, at least three genes, APP, PS1, and PS2, have been identified as causative genes. Mutations in these genes have been found to cause mainly early-onset AD. On the other hand, APOE has been identified to be the most common high genetic risk factor for late-onset AD. Polymorphisms in the coding region, intron, and promoter region of certain genes constitute another kind of genetic variation associated with AD. A number of other genes or loci have been reported to have linkage with AD, but many show only a weak linkage or the results are not well reproduced. Currently, the measurable genetic associations account for about 50% of the population risk for AD. It is believed that more new loci will be found to associate with AD, either as causative genes or genetic risk factors, and that eventually the understanding of genetic factors in the pathogenesis of AD will be important for our efforts to cure this illness.     

Binding of trypsin to fibrillar amyloid beta-protein

We have recently reported that fibrillar amyloid beta-protein (Abeta) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Abeta (fAbeta) and the resulting complex of trypsin/fAbeta is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Abeta 1-40 and Abeta 1-42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAbeta sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAbeta that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Abeta was dependent on the degree of Abeta fibrillization and on the concentration of fAbeta. However, the trypsin binding to Abeta oligomers did not affect the fibril growth. The maximum binding (B(max)) of trypsin to fAbeta 1-40 and fAbeta 1-42 was 36 pmol and 40 pmol, and dissociation constant (K(d)) was 18.31 microM and 20 microM respectively. Similar to fAbeta, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Abeta. These results suggest that fAbeta and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.