Zileuton restores memory impairments and reverses amyloid and tau pathology in aged Alzheimer's disease mice

The enzyme 5-lipoxygenase (5LO) is upregulated in Alzheimer's disease (AD), and its pharmacologic blockade with zileuton slows down the development of the AD-like phenotype in young AD mice. However, its efficacy after the AD pathology is established is unknown. To this end, starting at 12 months of age triple transgenic mice (3xTg) received zileuton, a selective 5LO inhibitor, or placebo for 3 months, and then the effect of this treatment on behavior, amyloid, and tau pathology assessed. Although mice on placebo showed worsening of their memory, treated mice performed even better than at baseline. Compared with placebo, treated mice had significantly less Aβ deposits and tau phosphorylation secondary to reduced γ-secretase and CDK-5 activation, respectively. Our data provide novel insights into the disease-modifying action of pharmacologically inhibiting 5LO as a viable AD therapeutic approach. They represent the successful completion of preclinical studies for the development of this class of drug as clinically applicable therapy for the disease.     

The role of synaptic activity in the regulation of amyloid beta levels in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia. Accumulation of amyloid-beta (Aβ) peptides is regarded as the critical component associated with AD pathogenesis, which is derived from the amyloid precursor protein (APP) cleavage. Recent studies suggest that synaptic activity is one of the most important factors that regulate Aβ levels. It has been found that synaptic activity facilitates APP internalization and influences APP cleavage. Glutamatergic, cholinergic, serotonergic, leptin, adrenergic, orexin, and gamma-amino butyric acid receptors, as well as the activity-regulated cytoskeleton-associated protein (Arc) are all involved in these processes. The present review summarizes the evidence for synaptic activity-modulated Aβ levels and the mechanisms underlying this regulation. Interestingly, the immediate early gene product Arc may also be the downstream signaling molecule of several receptors in the synaptic activity-modulated Aβ levels. Elucidating how Aβ levels are regulated by synaptic activity may provide new insights in both the understanding of the pathogenesis of AD and in the development of therapies to slow down the progression of AD.     

The normal equilibrium between CSF and plasma amyloid beta levels is disrupted in Alzheimer's disease

Amyloid-beta (Abeta) with 40 (Abeta40) and 42 (Abeta42) amino acids, the main components of amyloid plaques in the Alzheimer's disease (AD) brain, can be measured in human cerebrospinal fluid (CSF) and plasma. Whereas CSF Abeta42 is decreased in AD, some studies have reported changed plasma Abeta levels in AD and in subjects with mild cognitive impairment (MCI). To this date it is unclear if and how CSF and plasma levels of Abeta correlate with each other in healthy individuals, albeit earlier studies on AD patients found no correlation between CSF and plasma Abeta. We have measured Abeta40 and Abeta42 in paired CSF and plasma samples from patients with AD (n=39), MCI (n=29) and healthy control subjects (n=18). We observed a clear correlation between CSF and plasma levels for both Abeta40 and Abeta42 in healthy individuals, whereas no such correlation could be seen for AD or MCI cases. Similarly to other studies we also found low levels of Abeta42 in AD CSF, whereas there were no significant differences in plasma Abeta levels between the diagnostic groups. Our findings suggest that the normal equilibrium between CSF and plasma Abeta may be disrupted with the initiation of amyloid deposition in the brain.     

Accelerated tau pathology with synaptic and neuronal loss in a novel triple transgenic mouse model of Alzheimer's disease

There is pivotal evidence that tau pathology can be triggered by amyloid-β (Aβ) pathology in experimental systems. On the other side, studies on human brain specimen have elucidated that tau pathology may occur before amyloid pathology is present indicating that in principle tau pathology could also trigger Aβ aggregation. To address this question, we have crossed 5XFAD mice coexpressing human mutant APP695 with the Swedish, Florida, and London mutations and human mutant presenilin-1 (PS1) with the M146L and L286V mutations with the PS19 model overexpressing human mutant tau with the P301S mutation. The resulting triple transgenic model 5XFAD/PS19 has been characterized at 3 and 9 months of age. A dramatic aggravation of hyperphosphorylated tau pathology together with a dramatically increased inflammatory response and a loss of synapses and hippocampal CA1 neurons in aged 5XFAD/PS19 mice were observed. Extracellular amyloid deposits were unaltered. These data support the assumption of tau pathology being downstream of amyloid pathology, suggesting that both pathologies together trigger the severe neuron loss in the hippocampus in the 5XFAD/PS19 mouse model.     

Co-localization of amyloid beta and tau pathology in Alzheimer's disease synaptosomes

The amyloid cascade hypothesis proposes that amyloid beta (Abeta) pathology precedes and induces tau pathology, but the neuropathological connection between these two lesions has not been demonstrated. We examined the regional distribution and co-localization of Abeta and phosphorylated tau (p-tau) in synaptic terminals of Alzheimer's disease brains. To quantitatively examine large populations of individual synaptic terminals, flow cytometry was used to analyze synaptosomes prepared from cryopreserved Alzheimer's disease tissue. An average 68.4% of synaptic terminals in the Alzheimer's disease cohort (n = 11) were positive for Abeta, and 32.3% were positive for p-tau; Abeta and p-tau fluorescence was lowest in cerebellum. In contrast to synaptic p-tau, which was highest in the entorhinal cortex and hippocampus (P = 0.004), synaptic Abeta fluorescence was significantly lower in the entorhinal cortex and hippocampus relative to neocortical regions (P = 0.0003). Synaptic Abeta and p-tau fluorescence was significantly correlated (r = 0.683, P < 0.004), and dual-labeling experiments demonstrated that 24.1% of Abeta-positive terminals were also positive for p-tau, with the highest fraction of dual labeling (39.3%) in the earliest affected region, the entorhinal cortex. Western blotting experiments show a significant correlation between synaptic Abeta levels measured by flow cytometry and oligomeric Abeta species (P < 0.0001). These results showing overlapping Abeta and tau pathology are consistent with a model in which both synaptic loss and dysfunction are linked to a synaptic amyloid cascade within the synaptic compartment.     

Lithium down-regulates tau in cultured cortical neurons: a possible mechanism of neuroprotection

In tauopathies such as Alzheimer's disease (AD), the moleccular mechanisms of tau protein agregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration are not fully understood. Recent studies indirectly demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration, especially that induced by the amyloid (Abeta) pathology. Lithium is a medication for bipolar mood disorders. Its therapeutic mechanism of action remains unclear, in part because of the large number of biochemical effects attributed to lithium. Since lithium directly inhibits glycogen synthase kinase-3beta (GSK3beta), a key enzyme involved in tau phosphorylation, it was suggested that the therapeutic use of lithium could be expanded from mood disorders to neurodegenerative conditions. Lithium has been also reported to protect cultured neurons against Abeta toxicity, and to prevent NFTs accumulation and cognitive impairments in transgenic models of tauopathies. However, the exact mechanism of neuroprotection provided by lithium remains unknown. Here, we show that exposure of cultured cortical neurons to lithium decreased tau protein levels. This decrease was not linked to the activation of proteolytic processes including calpains, caspases and proteasome or to neuronal loss, but was rather associated with a reduction in tau mRNA levels. Moreover, prior exposure to lithium, at concentrations effective in reducing tau protein levels, markedly reduced pre-aggregated Abeta-induced neuronal apoptosis. Our findings raise the possibility that lithium could exert its neuroprotective effect against Abeta toxicity through the downregulation of tau proteins and that, at least, by acting at the level of tau mRNA.     

Knockout of plasminogen activator inhibitor 1 gene reduces amyloid beta peptide burden in a mouse model of Alzheimer's disease

Accumulation of amyloid beta peptide (Aβ) in the brain is a pathological hallmark of Alzheimer's disease (AD); the underlying mechanism, however, is not well understood. In this study, we show that expression of plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), increases with age in the brain of wild type and Aβ precursor protein-presenilin 1 (APP/PS1) transgenic mice as well as in AD patients. Most importantly, we show that knocking out the PAI-1 gene dramatically reduces Aβ burden in the brain of APP/PS1 mice but has no effect on the levels of full-length APP, alpha or beta C-terminal fragments. Furthermore, we show that knocking out the PAI-1 gene leads to increases in the activities of tPA and plasmin, and the plasmin activity inversely correlates with the amounts of SDS insoluble Aβ40 and Aβ42. Together, these data suggest that increased PAI-1 expression/activity contributes importantly to Aβ accumulation during aging and in AD probably by inhibiting plasminogen activation and thus Aβ degradation.     

On the origin of Alzheimer's disease. Trials and tribulations of the amyloid hypothesis

The amyloid cascade hypothesis, which implicates the amyloid Aβ peptide as the pathological initiator of both familial and sporadic, late onset Alzheimer's disease (AD), continues to guide the majority of research. We believe that current evidence does not support the amyloid cascade hypothesis for late onset AD. Instead, we propose that Aβ is a key regulator of brain homeostasis. During AD, while Aβ accumulation may occur in the long term in parallel with disease progression, it does not contribute to primary pathogenesis. This view predicts that amyloid-centric therapies will continue to fail, and that progress in developing successful alternative therapies for AD will be slow until closer attention is paid to understanding the physiological function of Aβ and its precursor protein.     

Gender effect on the accumulation of hyperphosphorylated tau in the brain of locus-ceruleus-injured APP-transgenic mouse

Locus ceruleus (LC) neurons are preferentially and initially affected in Alzheimer disease (AD); however, the impact of the loss of LC neurons on the pathological sequence of AD, including amyloid β-protein (Aβ) deposition and neurofibrillary tangle formation, has not been elucidated. In this study, we chemically injured LC neurons of the brains of familial AD-related amyloid precursor protein (APP)-transgenic mice using the LC-noradrenergic neuron-selective neurotoxin, N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (DSP4). The levels of noradrenaline significantly decreased in the cerebral cortices of DSP4-treated mice. The deposition of amyloid fibrils was biochemically observed in the APP-transgenic mouse brains; however, those levels were not significantly altered following DSP4 treatment. In contrast, the levels of accumulated hyperphosphorylated tau markedly increased in the cerebral cortices of DSP4-treated female but not male APP-transgenic mice. Our results suggest that innervation from LC neurons and testosterone secretion are potent and mutually independent suppressors of amyloid-related accumulation of hyperphosphorylated tau in the brain.     

Gadolinium-staining reveals amyloid plaques in the brain of Alzheimer's transgenic mice

Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimer's disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by μMRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.     

The role of intracellular amyloid beta in Alzheimer's disease

Extracellular amyloid beta (Abeta) that confers neurotoxicity and modulates synaptic plasticity and memory function has been central to the amyloid hypothesis of Alzheimer's disease (AD) pathology. Like many other misfolded proteins identified in neurodegenerative disorders, Abeta also accumulates inside the AD neurons. This intracellular Abeta affects a variety of cellular physiology from protein degradation, axonal transport, autophagy to apoptosis, further documenting the role of Abeta in AD. Therapeutics targeting intracellular Abeta could be effective treatment for AD.     

Differential recognition of vascular and parenchymal beta amyloid deposition

By phage display, llama-derived heavy chain antibody fragments were selected from non-immune and immune libraries and tested for their affinity and specificity for beta amyloid by phage-ELISA, immunohistochemistry and surface plasmon resonance.We identified eight distinct heavy chain antibody fragments specific for beta amyloid. While three of them recognized vascular and parenchymal beta amyloid deposits, the remaining five heavy chain antibody fragments recognized vascular beta amyloid specifically, failing to bind to parenchymal beta amyloid.These heavy chain antibody fragments, selected from different libraries, demonstrated differential affinity for different epitopes when used for immunohistochemistry. These observations indicate that the llama heavy chain antibody fragments are the first immunologic probes with the ability to differentiate between parenchymal and vascular beta amyloid aggregates. This indicates that vascular and parenchymal beta amyloid deposits are heterogeneous in epitope presence/availability. The properties of these heavy chain antibody fragments make them potential candidates for use in in vivo differential diagnosis of Alzheimer disease and cerebral amyloid angiopathy. Continued use and characterization of these reagents will be necessary to fully understand the performance of these immunoreagents.     

Diverse Molecular Targets for Therapeutic Strategies in Alzheimer's Disease

Alzheimer''s disease (AD) is the most common form of dementia caused by neurodegenerative process and is tightly related to amyloid β (Aβ) and neurofibrillary tangles. The lack of early diagnostic biomarker and therapeutic remedy hinders the prevention of increasing population of AD patients every year. In spite of accumulated scientific information, numerous clinical trials for candidate drug targets have failed to be preceded into therapeutic development, therefore, AD-related sufferers including patients and caregivers, are desperate to seek the solution. Also, effective AD intervention is desperately needed to reduce AD-related societal threats to public health. In this review, we summarize various drug targets and strategies in recent preclinical studies and clinical trials for AD therapy: Allopathic treatment, immunotherapy, Aβ production/aggregation modulator, tau-targeting therapy and metabolic targeting. Some has already failed in their clinical trials and the others are still in various stages of investigations, both of which give us valuable information for future research in AD therapeutic development.     

Mitochondrial DNA damage in a mouse model of Alzheimer's disease decreases amyloid beta plaque formation

Mitochondrial DNA (mtDNA) damage and the generation of reactive oxygen species have been associated with and implicated in the development and progression of Alzheimer's disease. To study how mtDNA damage affects reactive oxygen species and amyloid beta (Aβ) pathology in vivo, we generated an Alzheimer's disease mouse model expressing an inducible mitochondrial-targeted endonuclease (Mito-PstI) in the central nervous system. Mito-PstI cleaves mtDNA causing mostly an mtDNA depletion, which leads to a partial oxidative phosphorylation defect when expressed during a short period in adulthood. We found that a mild mitochondrial dysfunction in adult neurons did not exacerbate Aβ accumulation and decreased plaque pathology. Mito-PstI expression altered the cleavage pathway of amyloid precursor protein without increasing oxidative stress in the brain. These data suggest that mtDNA damage is not a primary cause of Aβ accumulation.     

Detection of amyloid beta protein in the urine of Alzheimer's disease patients and healthy individuals

To seek for a new valid biomarker using non-invasive specimens for the diagnosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI), we carried out the detection of amyloid beta (Abeta) protein in urine. Ten-millilitre urine samples were first sedimented with trichloroacetic acid, and the pellets were resuspended for further analysis by Western blotting with anti-Abeta antibody. The detection sensitivity of the method was 40pg/ml. Rates of subjects positive for monomeric Abeta according to their clinical dementia rating (CDR) were 11.1% for CDR 0, 62.5% for CDR 0.5, 83.3% for CDR 1, 54.5% for CDR 2 and 0% for CDR 3. A single Abeta band relative to the CDR score reflects an alteration in the production, solubility and clearance of Abeta in the brain. Thus, the method could be used as both a diagnostic and monitoring tool in assessing AD and MCI patients during disease-modifying therapies.     

Neuropep-1 ameliorates learning and memory deficits in an Alzheimer's disease mouse model,increases brain-derived neurotrophic factor expression in the brain,and causes reduction of amyloid beta plaques

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aβ) deposits, hyperphosphorylated tau deposition, and cognitive dysfunction. Abnormalities in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in learning and memory formation, have been reported in the brains of AD patients. A BDNF modulating peptide (Neuropep-1) was previously identified by positional-scanning synthetic peptide combinatorial library. Here we examine the neuroprotective effects of Neuropep-1 on several in vitro neurotoxic insults, and triple-transgenic AD mouse model (3xTg-AD). Neuropep-1 protects cultured neurons against oligomeric Aβ_1–42, 1-methyl-4-phenylpyridinium, and glutamate-induced neuronal cell death. Neuropep-1 injection also significantly rescues the spatial learning and memory deficits of 3xTg-AD mice compared with vehicle-treated control group. Neuropep-1 treatment markedly increases hippocampal and cortical BDNF levels. Furthermore, we found that Neuropep-1-injected 3xTg-AD mice exhibit dramatically reduced Aβ plaque deposition and Aβ levels without affecting tau pathology. Neuropep-1 treatment does not alter the expression or activity of full-length amyloid precursor protein, α-, β-, or γ-secretase, but levels of insulin degrading enzyme, an Aβ degrading enzyme, were increased. These findings suggest Neuropep-1 may be a therapeutic candidate for the treatment of AD.     

Pathological relationships between microglial cell activity and tau and amyloid beta protein in patients with Alzheimer's disease

The present study directly examined the axon terminal (synaptosomal) level of amino acid neurotransmitters in rats following chronic constriction injury (CCI) to the sciatic nerve. Dorsal horn, synaptosomal content of aspartate (Asp), glutamate (Glu), glycine and gamma-aminobutyric acid (GABA) was assessed in these rats, and in normal age-matched and younger rats. The synaptosomal content of Asp and Glu in CCI rats was increased by 44-46% compared with control rats (P<0.016). The synaptosomal content of GABA in younger rats was 33% lower than that observed in control rats (P<0.005). Altered axon terminal levels of amino acid neurotransmitters accompany the painful symptoms of neuropathy. The lower axon terminal level of GABA in younger rats may help to explain the age-dependency of pain development in animal models of nerve injury.     

Fractal analysis of amyloid plaques in Alzheimer's disease patients and mouse models

The varied morphological and biochemical forms in which amyloid deposits in brain of Alzheimer's disease (AD) patients are complex and their mechanisms of formation are not completely understood. Here we investigated the ability of fractal dimension (FD) to differentiate between the textures of commonly observed amyloid plaques in sporadic and familial AD patients and aged-control individuals as well as in transgenic mouse models of amyloidosis. Studying more than 6000 amyloid plaques immunostained for total Aβ (Aβt), Aβ40 or Aβ42, we show here that Aβ40 FD could efficiently differentiate between (i) AD patients and aged-control individuals (P < 0.001); (ii) sporadic and familial AD due to presenilin-1 or APP (A692G) mutations (P < 0.001); and (iii) three transgenic mouse models of different genotypes (P < 0.001). Furthermore, while diffuse and dense-core plaques present in humans and transgenic mice had comparable FDs, both Aβt and Aβ42 FD could also differentiate diffuse plaques from other plaque types in both species (P < 0.001). Our data suggest that plaque FD could be a valuable tool for objective, computer-oriented AD diagnosis as well as for genotype-phenotype correlations of AD.     

Endolysosome involvement in HIV-1 transactivator protein-induced neuronal amyloid beta production

The increased life expectancy of people living with HIV-1/AIDS is accompanied by increased prevalence of HIV–1-associated neurocognitive disorder. As well, these individuals are increasingly experiencing Alzheimer's disease (AD)-like neurocognitive problems and neuropathological features such as increased deposition of amyloid beta (Aβ) protein. Findings that Aβ production occurs largely in endolysosomes, that HIV-1 transactivator protein (Tat) disrupts endolysosome function—an early pathological feature of AD—and that HIV-1 Tat can increase Aβ levels prompted us to test the hypothesis that endolysosome dysfunction is associated with HIV-1 Tat-induced increases in neuronal Aβ generation. Using primary cultured rat hippocampal neurons, we found that treatment with HIV-1 Tat caused such morphological changes as enlargement of endolysosomes identified with LysoTracker dye and such functional changes as elevated endolysosome pH measured ratiometrically with LysoSensor dye. The HIV-1 Tat-induced changes in endolysosome function preceded temporally HIV-1 Tat-induced increases in Aβ generation measured using enzyme-linked immunosorbent assay. In addition, we demonstrated that HIV-1 Tat increased endolysosome accumulation of Aβ precursor protein and Aβ identified using immunostaining with 4G8 antibodies. Furthermore, we demonstrated that treatment of neurons with HIV-1 Tat increased endolysosome accumulation of beta amyloid-converting enzyme, the rate-limiting enzymatic step for Aβ production, and enhanced beta amyloid-converting enzyme activity. Together, our findings suggest that HIV-1 Tat increases neuronal Aβ generation and thereby contributes to the development of AD-like pathology in HIV–1-infected individuals by disturbing endolysosome structure and function.