“Amyloid‐beta accumulation cycle” as a prevention and/or therapy target for Alzheimer's disease

The cell cycle and its regulators are validated targets for cancer drugs. Reagents that target cells in a specific cell cycle phase (e.g., antimitotics or DNA synthesis inhibitors/replication stress inducers) have demonstrated success as broad‐spectrum anticancer drugs. Cyclin‐dependent kinases (CDKs) are drivers of cell cycle transitions. A CDK inhibitor, flavopiridol/alvocidib, is an FDA‐approved drug for acute myeloid leukemia. Alzheimer's disease (AD) is another serious issue in contemporary medicine. The cause of AD remains elusive, although a critical role of latent amyloid‐beta accumulation has emerged. Existing AD drug research and development targets include amyloid, amyloid metabolism/catabolism, tau, inflammation, cholesterol, the cholinergic system, and other neurotransmitters. However, none have been validated as therapeutically effective targets. Recent reports from AD‐omics and preclinical animal models provided data supporting the long‐standing notion that cell cycle progression and/or mitosis may be a valid target for AD prevention and/or therapy. This review will summarize the recent developments in AD research: (a) Mitotic re‐entry, leading to the “amyloid‐beta accumulation cycle,” may be a prerequisite for amyloid‐beta accumulation and AD pathology development; (b) AD‐associated pathogens can cause cell cycle errors; (c) thirteen among 37 human AD genetic risk genes may be functionally involved in the cell cycle and/or mitosis; and (d) preclinical AD mouse models treated with CDK inhibitor showed improvements in cognitive/behavioral symptoms. If the “amyloid‐beta accumulation cycle is an AD drug target” concept is proven, repurposing of cancer drugs may emerge as a new, fast‐track approach for AD management in the clinic setting.     

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.     

Mouse models of Alzheimer's disease: a quest for plaques and tangles

Many genetically altered mice have been designed to help understand the role of specific gene mutations in the pathogenesis of Alzheimer's disease (AD) based on the realization that specific mutations in the genes for amyloid precursor protein--the presenilins and tau--are associated with early-onset familial AD or, in the case of tau mutations, other neurodegenerative diseases with neurofibrillary tangles. However, attempts to reproduce the neuropathology of AD in the mouse have been frustrating. Transgenic designs emphasizing amyloid precursor protein produced mice that develop amyloid plaques, but neurodegeneration and neurofibrillary tangles failed to form. Strategies emphasizing tau resulted in increased phosphorylation of tau and tangle formation, although amyloid plaques were absent. Nevertheless, crossing transgenic animals expressing mutated tau and amyloid precursor protein has produced a mouse that closely recapitulates the neuropathology of AD. A review of the various murine models, their role in understanding the pathogenesis of AD and their use in testing therapeutic regimens, is provided.     

Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.     

Amyloid PET: Its diagnostic potential compared to FDG

Amyloid PET using high-affinity ligands for fibrillary amyloid is providing high specificity and sensitivity for detection of Alzheimer's disease (AD) even before onset of dementia. Most current published data have been acquired using 11C-Pittsburgh Compound B (PIB). However, due to the extremely short half-life of 11C, PIB is available only in some research laboratories. This limitation will be overcome by 18F-labeled ligands which are currently undergoing formal clinical trials as amyloid imaging agents and are expected to become commercially available for clinical use in the near future. Compared to FDG, which demonstrates regional metabolic deficits in AD and late-stage mild cognitive impairment (MCI), amyloid imaging is expected to provide higher sensitivity for early detection of AD. By detecting amyloid, it is providing information that is complementary to clinical symptoms, while FDG-PET is more closely related to dementia severity and cognitive symptoms. Current data suggest that a negative amyloid PET scan is likely to rule out AD with more than 90% certainty, while a positive scan in a dementia patient or a patient with amnestic MCI indicates a very high likelihood of AD. There is still uncertainty about the clinical significance of positive amyloid scans in elderly normal controls (10 to 40% depending on age and selection criteria).     

The 717Val----Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.     

Morphological and pathological evolution of the brain microcirculation in aging and Alzheimer's disease

Key pathological hallmarks of Alzheimer's disease (AD), including amyloid plaques, cerebral amyloid angiopathy (CAA) and neurofibrillary tangles do not completely account for cognitive impairment, therefore other factors such as cardiovascular and cerebrovascular pathologies, may contribute to AD. In order to elucidate the microvascular changes that contribute to aging and disease, direct neuropathological staining and immunohistochemistry, were used to quantify the structural integrity of the microvasculature and its innervation in three oldest-old cohorts: 1) nonagenarians with AD and a high amyloid plaque load; 2) nonagenarians with no dementia and a high amyloid plaque load; 3) nonagenarians without dementia or amyloid plaques. In addition, a non-demented (ND) group (average age 71 years) with no amyloid plaques was included for comparison. While gray matter thickness and overall brain mass were reduced in AD compared to ND control groups, overall capillary density was not different. However, degenerated string capillaries were elevated in AD, potentially suggesting greater microvascular "dysfunction" compared to ND groups. Intriguingly, apolipoprotein ε4 carriers had significantly higher string vessel counts relative to non-ε4 carriers. Taken together, these data suggest a concomitant loss of functional capillaries and brain volume in AD subjects. We also demonstrated a trend of decreasing vesicular acetylcholine transporter staining, a marker of cortical cholinergic afferents that contribute to arteriolar vasoregulation, in AD compared to ND control groups, suggesting impaired control of vasodilation in AD subjects. In addition, tyrosine hydroxylase, a marker of noradrenergic vascular innervation, was reduced which may also contribute to a loss of control of vasoconstriction. The data highlight the importance of the brain microcirculation in the pathogenesis and evolution of AD.     

Comparative analysis of amyloid-beta chemical structure and amyloid plaque morphology of transgenic mouse and Alzheimer's disease brains

We have undertaken an integrated chemical and morphological comparison of the amyloid-beta (Abeta) molecules and the amyloid plaques present in the brains of APP23 transgenic (tg) mice and human Alzheimer's disease (AD) patients. Despite an apparent overall structural resemblance to AD pathology, our detailed chemical analyses revealed that although the amyloid plaques characteristic of AD contain cores that are highly resistant to chemical and physical disruption, the tg mice produced amyloid cores that were completely soluble in buffers containing SDS. Abeta chemical alterations account for the extreme stability of AD plaque core amyloid. The corresponding lack of post-translational modifications such as N-terminal degradation, isomerization, racemization, pyroglutamyl formation, oxidation, and covalently linked dimers in tg mouse Abeta provides an explanation for the differences in solubility between human AD and the APP23 tg mouse plaques. We hypothesize either that insufficient time is available for Abeta structural modifications or that the complex species-specific environment of the human disease is not precisely replicated in the tg mice. The appraisal of therapeutic agents or protocols in these animal models must be judged in the context of the lack of complete equivalence between the transgenic mouse plaques and the human AD lesions.     

Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Aβ) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Aβ specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Aβ oligomers (∼ > 60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Aβ oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Aβ channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.     

Identification and biology of α-secretase

Our knowledge of the etiology of Alzheimer's disease (AD) has advanced tremendously since the discovery of amyloid beta (Aβ) aggregation in diseased brains. Accumulating evidence suggests that Aβ plays a causative role in AD. The β-secretase enzyme, beta-site APP cleaving enzyme-1 (BACE1), is also implicated in AD pathogenesis, given that BACE1 cleavage of amyloid precursor protein is the initiating step in the formation of Aβ. As a result, BACE1 inhibition has been branded as a potential AD therapy. In this study, we review the identification and basic characteristics of BACE1, as well as the progress in our understanding of BACE1 cell biology, substrates, and phenotypes of BACE1 knockout mice that are informative about the physiological functions of BACE1 beyond amyloid precursor protein cleavage. These data are crucial for predicting potential mechanism-based toxicity that would arise from inhibiting BACE1 for the treatment or prevention of AD.     

Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion

Several lines of evidence suggest that dysregulated lipid metabolism may participate in the pathogenesis of Alzheimer’s disease (AD). Epidemiologic studies suggest that elevated mid-life plasma cholesterol levels may be associated with an increased risk of AD and that statin use may reduce the prevalence of AD. Cellular studies have shown that the levels and distribution of intracellular cholesterol markedly affect the processing of amyloid precursor protein into Aβ peptides, which are the toxic species that accumulate as amyloid plaques in the AD brain. Most importantly, genetic evidence identifies apolipoprotein E, the major cholesterol carrier in the central nervous system, as the primary genetic risk factor for sporadic AD. In humans, apoE exists as three major alleles (apoE2, apoE3, and apoE4), and inheritance of the apoE4 allele increases the risk of developing AD at an earlier age. However, exactly how apoE functions in the pathogenesis of AD remains to be fully determined. Our studies have identified that the cholesterol transporter ABCA1 is a crucial regulator of apoE levels and lipidation in the brain. Deficiency of ABCA1 leads to the loss of approximately 80% of apoE in the brain, and the residual 20% that remains is poorly lipidated. Several independent studies have shown this poorly lipidated apoE increases amyloid burden in mouse models of AD, demonstrating that apoE lipidation by ABCA1 affects key steps in amyloid deposition or clearance. Conversely, robust overexpression of ABCA1 in the brain promotes apoE lipidation and nearly eliminates the formation of mature amyloid plaques. These studies show that the lipid binding capacity of apoE is a major mechanism of its function in the pathogenesis of AD, and suggest that increasing apoE lipidation may be of therapeutic importance for this devastating disease.     

Biochemical detection of Abeta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer's disease

Prior to the identification of the various abnormal proteins deposited as fibrillar aggregates in the Alzheimer's disease (AD) brain, there was tremendous controversy over the importance of the various lesions with respect to primacy in the pathology of AD. Nevertheless, based on analogy to systemic amyloidosis, many investigators believed that the amyloid deposits in AD played a causal role and that characterization of these deposits would hold the key to understanding this complex disease. Indeed, in retrospect, it was the initial biochemical purifications of the approximately 4 kDa amyloid beta-peptide (Abeta) from amyloid deposits in the mid 1980s that launched a new era of AD research (Glenner and Wong, Biochem. Biophys. Res. Commun. 122 (1984) 1121-1135; Wong et al., Proc. Natl. Acad Sci. USA 82 (1985) 8729 8732; and Masters et al., Proc. Natl. Acad Sci. USA 82 (1985) 4245-4249). Subsequent studies of the biology of Abeta together with genetic studies of AD have all supported the hypothesis that altered Abeta metabolism leading to aggregation plays a causal role in AD. Although there remains controversy as to whether Abeta deposited as classic amyloid or a smaller, aggregated, form causes AD, the relevance of studying the amyloid deposits has certainly been proven. Despite the significant advances in our understanding of the role of Abeta in AD pathogenesis, many important aspects of Abeta biology remain a mystery. This review will highlight those aspects of Abeta biology that have led to our increased understanding of the pathogenesis of AD as well as areas which warrant additional study.     

Amyloid beta-protein gene duplication is not common in Alzheimer's disease: analysis by polymorphic restriction fragments

The amyloid beta-protein(BP) is an important component of amyloid fibrils of both Alzheimer's disease(AD) and adult Down syndrome(DS). It has been hypothesized that sporadic AD may involve the duplication of a subregion of chromosome 21 containing the BP locus. However, an improved method for detection of the BP gene duplication using polymorphic Hind III fragments led us to a conclusion that BP gene duplication is rare, if any, in (Japanese) sporadic AD patients, indicating that the duplication of the BP gene itself is not the common underlying genetic defect in AD.     

Tau PET Imaging for Staging of Alzheimer's Disease in Down Syndrome

Alzheimer's disease (AD) pathology and early‐onset dementia develop almost universally in Down syndrome (DS). AD is defined neuropathologically by the presence of extracellular plaques of aggregated amyloid β protein and intracellular neurofibrillary tangles (NFTs) of aggregated hyperphosphorylated tau protein. The development of radiolabeled positron emission tomography (PET) ligands for amyloid plaques and tau tangles enables the longitudinal assessment of the spatial pattern of their accumulation in relation to symptomatology. Recent work indicates that amyloid pathology develops 15–20 years before neurodegeneration and symptom onset in the sporadic and autosomal dominant forms of AD, while tau pathology correlates more closely with symptomatic stages evidenced by cognitive decline and dementia. Recent work on AD biomarkers in DS illustrates similarities between DS and sporadic AD. It may soon be possible to apply recently developed staging classifications to DS to obtain a more nuanced understanding of the development AD in DS and to provide more accurate diagnosis and prognosis in the clinic.     

Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice

Trafficking through the secretory pathway is known to regulate the maturation of the APP-cleaving secretases and APP proteolysis. The coupling of stress signaling and pathological deterioration of the brain in Alzheimer's disease (AD) supports a mechanistic connection between endoplasmic reticulum (ER) stress and neurodegeneration. Consequently, small molecular chaperones, which promote protein folding and minimize ER stress, might be effective in delaying or attenuating the deleterious progression of AD. We tested this hypothesis by treating APPswePS1delta9 AD transgenic mice with the molecular chaperone phenylbutyric acid (PBA) for 14 months at a dose of 1 mg PBA g(-1) of body weight in the drinking water. Phenylbutyric acid treatment increased secretase-mediated APP cleavage, but was not associated with any increase in amyloid biosynthesis. The PBA-treated AD transgenic mice had significantly decreased incidence and size of amyloid plaques throughout the cortex and hippocampus. There was no change in total amyloid levels suggesting that PBA modifies amyloid aggregation or pathogenesis independently of biogenesis. The decrease in amyloid plaques was paralleled by increased memory retention, as PBA treatment facilitated cognitive performance in a spatial memory task in both wild-type and AD transgenic mice. The molecular mechanism underlying the cognitive facilitation of PBA is not clear; however, increased levels of both metabotropic and ionotropic glutamate receptors, as well as ADAM10 and TACE, were observed in the cortex and hippocampus of PBA-treated mice. The data suggest that PBA ameliorates the cognitive and pathological features of AD and supports the investigation of PBA as a therapeutic for AD.     

Neuroimaging Results Impose New Views on Alzheimer’s Disease—the Role of Amyloid Revised

Huge progress has been made in unraveling the mysteries of Alzheimer’s disease (AD), but we still do not understand the basic mechanisms that set off the cascade of pathological events. In May 2011, the National Institute on Aging–Alzheimer’s Association published new diagnostic guidelines, expected to have huge impact on AD research and clinical practice. However, the new guidelines are already criticized for being biased in favor of a specific theory of the pathophysiological origins of AD—the amyloid cascade hypothesis. Shortly before publication of the guidelines, a hypothetical model of the dynamic biomarkers of the Alzheimer’s pathological cascade was published, taking as starting point that biomarkers reflecting brain levels of amyloid become deviant long before brain atrophy, cognitive dysfunction, or clinical symptoms are manifest. This model has already attracted substantial interest and arguably represents a dominating view within human research on AD. Here we critically review the evidence for the view of amyloid as an initiating event in the pathological cascade and discuss how central assumptions of this hypothesis affect how results from contemporary human AD research are understood. Interpretations of new results are greatly impacted by researchers’ view on the role of amyloid, and identical observations are sometimes taken to support radically opposing views on the amyloid hypothesis. We argue that the canonical view of the role of amyloid as the main causal factor in AD may not be correct and that evidence from recent neuroimaging studies indicates that amyloid is neither necessary nor sufficient, for the manifestation of AD-like brain atrophy.     

Time sequence of oxidative stress in the brain from transgenic mouse models of Alzheimer's disease related to the amyloid-β cascade

Alzheimer's disease (AD) is a multifactorial disorder characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs). Rare early-onset forms of AD are associated with autosomal dominant mutations in the amyloid precursor protein gene, presenilin 1 gene, or presenilin 2 gene. The late-onset form of the disease (LOAD) is the most common form. The causes of LOAD are not yet clarified, but several environmental and genetic risk factors have been identified. Numerous studies have highlighted a role for free radical-mediated injury to brain regions of this illness. In addition, studies from mild cognitive impairment patients suggest that oxidative stress is an early event in the pathogenesis of AD. The associations between these markers of free radical damage and the pathogenic cascades involved in AD are complex. Over the past 2 decades, a number of mouse models have been created to recapitulate the major neuropathological hallmarks of AD, namely amyloid plaques and NFTs. These mice recapitulate many, although not all, of the key features of AD. Some strains of transgenic mice develop amyloid plaques, some accumulate NFTs, and some do both. Here we review the evidence for increased free radical-mediated damage to the brain with particular attention to the stage of the disease in various transgenic models of AD related to the amyloid-β cascade.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)