Aggregating interests in aggregates: mechanistic, diagnostic and therapeutic implications of brain amyloidosis in neurodegenerative disorders among the elderly (2005)

Extensive neuronal damage provoked by the accumulation of amyloidogenic molecules is a central event in the pathological cascade of Alzheimer's disease (AD), Parkinson's disease (PD) and other related neuropsychiatric disorders. Genetic mutations that are directly related to the abnormalities of these pathogenic components result in familial forms of neurodegenerative diseases, while causal links between amyloidogenesis and neuronal injury in sporadic illnesses are yet to be pursued. A longitudinal in vivo investigation of brain amyloid deposition and nervous system degeneration in a trial for anti-amyloidogenic treatment may provide explicit evidence of the mechanistic contribution of amyloid aggregates to neurotoxicity. Blood–brain barrier-penetrating ligands for β-sheet fibrils offer visualization of extracellular and intracellular amyloid lesions in brains of living animal models and human subjects. As fibrillar deposits start piling up early in the pathogenesis of neurodegenerative conditions, the appearance of brain amyloids is likely to herald the clinical onset of disease. Moreover, a subset of β-sheet-binding compounds can arrest the fibrillization of amyloidogenic molecules, thereby enabling us to assess the impact of self-assembled fibrils on neuronal disintegration. It is thus conceivable that such amyloid antagonists will also be major role players in disease-modifying therapies for devastating neurodegenerative disorders. Amyloid deposits comprised of β-pleated sheet proteins and peptides are viewed as pathological hallmarks in the brains of elderly people with diverse neurodegenerative disorders.1 Extracellular senile plaques and intracellular neurofibrillary lesions, the major constituents of which are amyloid-β peptides and tau proteins, respectively, are amyloid fibrils and are consistently present in AD of all etiologies. Dementia with Lewy bodies (DLB), which is the second most common form of neurodegenerative dementia following AD, is neuropathologically characterized by the accumulation of intraneuronal α-synuclein fibrils called Lewy bodies and Lewy neurites. Moreover, fibrillary tau aggregates in neurons and glia define a distinct group of presenile/senile dementias, including argyrophilic grain disease and a subset of frontotemporal lobar degeneration (FTLD), collectively called tauopathies.2 Clinical manifestations of these illnesses predominantly involve cognitive domains, while brain amyloidosis is also implicated in neurological conditions primarily related to motor dysfunctions, such as PD and multiple system atrophy (MSA). Indeed, the close link between PD and DLB is illustrated by the presence of α-synuclein-positive Lewy bodies and Lewy neurites in both diseases, and degenerating neurons and glia in MSA are also overwhelmed by α-synuclein deposits.1 Since the 1990s, genetic mutations of these amyloidogenic molecules in related familial neurodegenerative diseases have been discovered and have provided evidence of causal relationships between the aberrant accumulation of brain amyloids and the onset of nervous system disorders. The mutation of genes coding amyloid precursor protein (APP) and presenilins lead to familial AD (FAD) primarily via the overproduction of Aβ.3 Mutant tau proteins either self-assemble at an accelerated rate or are defective in stabilizing axonal cytoskeletons, giving rise to a hereditary form of FTLD referred to as frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17).2 Missence mutations on the α-synuclein gene have been found in people with PD, and multiplication of the same gene has been shown to cause PD and DLB in a small subpopulation of familial cases.4 It is noteworthy that different amyloid pathologies frequently coexist in a single disease.1 The presence of fibrillary tau lesions in the brains of people with DLB, PD and MSA and α-synuclein aggregates in people with AD and FTLD implies that triple- or double-brain amyloidosis is characteristic of the vast majority of neurodegenerative diseases among the elderly. This also raises the point that the ‘pure’ mechanistic contribution of a single amyloidogenic molecule to neuronal injury may hardly be exclusive because of the concomitance of other amyloid pathologies. The difficulty of elucidating the molecular mechanism by which each type of amyloid lesion induces neurodegeneration provides a rationale for the investigation of genetically engineered animal models that develop single-brain amyloidosis. Recent transgenic technology has enabled the establishment of murine strains mimicking the amyloid deposition seen in human brains.1 Transgenic mice overexpressing FAD mutant APP with or without FAD mutant presenilin exhibit Aβ plaques in an age-dependent fashion, and progressive accumulation of neurofibrillary tangles is observed in mice transgenic in FTDP-17 mutant tau. Lewy body-like cytoplasmic inclusions are also found in familial PD mutant α-synuclein transgenic mice. Data obtained from experiments using these mouse models have revealed that the abnormality of amyloidogenic molecules deteriorates the integrity of neurons and glia via the loss of normal functions and increase in cytotoxicity. Although there is evidence to show that filamentous aggregates have a great impact on cellular structures and functions as compared to monomeric species, molecular assemblies at the initial stages of fibrillization are believed to be more toxic than mature amyloids. In fact, animal studies have shown that synaptic plasticity is vulnerable to Aβ oligomers,5 and that prominent neuronal loss occurs prior to the emergence of intracellular amyloid deposits composed of tau and α-synuclein that are visible at a light microscopic level.6 In addition to providing an insight into the critical effects of a single amyloidosis, genetically altered animals have been used to clarify the synergistic interactions between amyloidogenic molecules in the pathogenesis of neuropsychiatric conditions. The accelerated formation of tau and α-synuclein aggregates caused by Aβ pathology has been observed by cross-breeding APP transgenic mice with tau and α-synuclein transgenic mice,7,8 and it has been shown that tau and α-synuclein facilitate fibrillogenesis of each other in mice that are doubly transgenic for these molecules.9 The possible mechanisms governing this synergism include the seeding effects of a small amyloid mass that attracts different amyloidogenic proteins into the same intracellular compartment. This may provide a plausible explanation for the co-aggregation of tau and α-synuclein in a single inclusion, while more indirect links between pathogenic proteins/peptides, such as perturbed metabolism and trafficking of one molecule secondary to abnormal accumulation of other molecules, may also mediate the deposition of Aβ, tau and α-synuclein fibrils in distinct intracellular and extracellular locations. Basic research employing genetically engineered mice has so far provided an insight into the pathophysiological significance of amyloid fibrillogenesis in the devastating effects of AD and other degenerative illnesses. However, the extent of the toxic impact on neurons from pathological fibrils alone remains unclear, because the above-mentioned experimental paradigms are unable to dissociate polymeric fibril-induced cytotoxicity from the impaired functions of normal monomeric molecules. Such elucidation could be achieved by time-course monitoring of amyloid formation and neurodegeneration after amyloidogenesis has been stopped by therapeutic approaches. If there is only a weak mechanistic association between amyloid deposition and neuronal damage, suppression of fibrillization is unlikely to arrest the neuropathological cascade, and therefore a marked discrepancy between amyloid accumulation and neuronal dysfunction/loss should be observed following treatment. Information about this discrepancy can be gathered by conducting time-course studies; however, three requirements have to be satisfied: (i) quantification of amyloid levels in living brains; (ii) in vivo assessment of central nervous system (CNS) degeneration; and (iii) access to agents that are capable of inhibiting the polymerization of amyloidogenic molecules. The non-invasive monitoring of brain amyloids and neuronal injuries may be carried out by the use of imaging techniques to quantitatively visualize these pathological alterations, which would greatly contribute to diagnostic progress for human subjects. Anti-amyloid agents would also be a prime focus in the development of therapeutic approaches to neurodegenerative disorders, if a close association between brain amyloids and neuronal deficits is successfully proven. Numerous investigations have attempted to detect brain amyloids by using imaging technologies, mostly with the assistance of specific probes. Because the β-pleated sheet is a secondary structure commonly shared by amyloid lesions, chemicals that bind to β-sheets would make good candidates for a tracer compound. Among such chemicals, Congo red and thioflavin-T are well-known dyes that stain a wide range of amyloid pathologies. However, these compounds are not applied as intravenously administrable tracers for amyloid imaging because they do not pass the blood–brain barrier (BBB). Thus, smaller and/or uncharged derivatives of Congo red and thioflavin-T have been developed for BBB permeability, and radiolabeled versions of these amyloid ligands are used for positron emission tomography (PET) and single-photon emission computed tomography (SPECT).10,11 Although some of the radiotracers have been shown to be useful for differentiating AD patients from normal subjects in pilot studies, experimental data have not provided unequivocal evidence that these radiolabeled compounds administered at an imaging dose bind specifically to brain amyloids. Extracellular Aβ plaques, which appear to be the most accessible target in tracer-assisted amyloid imaging, have only been marginally captured in PET analyzes of APP transgenic mouse brains,12 unlike the abundant accumulation of the amyloid-binding tracer in AD brains. Hence, it still remains likely that hydrophobic compounds designed for transfer through the BBB are trapped by non-specific pathologies in human subjects, such as the accumulation of lipid within the vessel wall during atherogenesis. Visualization of individual Aβ plaques in living mouse models is enabled by optical imaging and magnetic resonance imaging (MRI) following parenteral administration of non-radioactive amyloid-binding compounds at a relatively high dose. Optical observation of parenchymal amyloid deposits is possible through the use of either two-photon fluorescence microscopy or the combination of a wide-field camera and fluorescent tracers with an emission wavelength covering the near-infrared range.13,14 Although the latter can yield a large field of view, the spatial resolution hardly allows the imaging of isolated plaques. The penetration of optical signals, which is confined to a few centimeters within biological tissues, also indicates that this technique may not be applicable to the imaging of living human brains. High-field, high-resolution MRI is suitable for isolating large plaques (50–100 µm in diameter) from surrounding tissues. Amyloid plaques can be detected by plain MRI without a contrast agent presumably because of the presence of iron within the lesions,15 while more specific plaque detection is achieved by contrast-enhanced MR technology using amyloid-binding molecules conjugated with paramagnetic compounds.16 Furthermore, fluorine-MRI is able to map the distribution of the amyloid probe containing fluorine with little background signal.17 These MR techniques are potentially applicable to human studies, and high-field MRI systems for humans would offer a means of visualizing Aβ plaques for the diagnosis of AD. However, the MRI of amyloid ligands at present is not sensitive enough to detect plaques as well as PET and SPECT, and therefore technological innovations as well as improvement of the tracer compounds are definitely needed when using MRI as a diagnostic tool for patients. In vivo detection of intraneuronal amyloids might not be readily achieved, as intravenously injected tracers need to be transferred through the BBB and subsequently through the neuronal cell membrane. Our preliminary data suggest that several amyloid-binding probes can bind to neurofibrillary tangles in tau transgenic mice after being intravenously infused. The tracers tested so far show affinity for both plaques and tangles, and thus do not differentiate between these two pathologies, while subtle conformational differences among Aβ, tau and α-synuclein fibrils may allow the exploitation of a ligand with a selectively high affinity for a certain form of fibrillar aggregate. A series of PET or SPECT scans with a combination of these tracers would facilitate the discrimination between AD and other tauopathies or α-synucleinopathies. Another potentially interesting property of the β-sheet ligands is that some compounds might exhibit a high affinity for the immature form of amyloid filaments relative to the binding to mature fibrils, giving rise to the possibility of imaging an early stage of amyloid formation. This could be of particular importance in the preclinical diagnosis of neurodegenerative disorders, and in the detection of oligomers and protofibrils that are more neurotoxic in their immature forms. Degenerative changes in the CNS are imaged as brain atrophy resulting from neuronal loss. However, neuron death is a terminal point of cytotoxic events, and accordingly, early neurotoxic injuries may not be accurately analyzed by morphometric imaging. Functional alterations, including reduced energy metabolism and compromised neurotransmission, precede neuronal loss and can be non-invasively monitored by PET. In addition, we have recently shown that a radioligand with a high affinity for peripheral benzodiazepine receptors facilitates the PET imaging of gliotic changes in living brains.18 Because pronounced neuronal death is frequently heralded by microgliosis and astrocytosis in pathological conditions, in vivo analysis of glial activation in a quantitative manner could be a powerful means for the precise assessment of neuroglial degeneration with sufficient sensitivity. A critical notion associated with amyloid-binding probes is that such compounds bound on preassembled fibrils are likely to inhibit further association of amyloidogenic components with the fibrils, and consequently suppress maturation of the filamentous aggregates when administered at a sufficiently high dose. Earlier investigations have explicitly indicated the inhibition of Aβ fibrillization by Congo red,19 and more recently, arrays of chemicals have been shown to disturb the assembly of Aβ and tau filaments.20 Although the evidence is primarily based on in vitro experiments without serious consideration of drug delivery through the BBB, the potential of a BBB-permeable Congo-red derivative for the treatment of brain amyloidosis has also been suggested in a study of prion protein aggregation using mouse models.21 While the preferable pharmacokinetic properties of tracer ligands for high-contrast amyloid imaging include the fast clearance of amyloid-unbound tracers from the brain tissue and a low affinity for non-specific binding sites, the long retention time of the compounds in the brain is conceivably required for anti-amyloid treatment, and the extent of non-specific binding might not be a crucial concern for this therapeutic purpose. The generation of amyloid lesions could be halted by chemicals that bind to mature β-sheet fibrils. However, the same treatment might possibly result in an increase of less polymerized and more neurotoxic forms of amyloidogenic molecules, leading to the augmentation of degenerative pathology. In the case of Aβ pathogenesis, this potential problem can be circumvented by simultaneously facilitating the degradation of monomeric and oligomeric species of Aβ. It has been reported that the neutral endopeptidase, neprilysin, is capable of degrading Aβ monomers and oligomers in the brain and can be up-regulated by stimulating receptors for neuropeptides, such as somatostatin.22 Hence, the use of β-sheet ligands in conjunction with an ‘up-regulator’ of neprilysin would suppress most stages of Aβ amyloidogenesis. In summary, the development of amyloid-binding compounds would lead to the pathology-specific diagnosis and treatment of AD, PD and other neurodegenerative disorders. A time-course experiment using these compounds in combination with animal models could reveal the mechanistic relationships between amyloid formation and CNS degeneration, and the clinical application of anti-amyloid treatment would determine the real impact of fibrillar aggregation on memory and other cognitive functions in the human brain.