Extracellular chaperones modulate the effects of Alzheimer’s patient cerebrospinal fluid on Aβ<Subscript>1-42</Subscript> toxicity and uptake

Alzheimer’s disease is characterised by the inappropriate death of brain cells and accumulation of the Aβ peptide in the brain. Thus, it is possible that there are fundamental differences between Alzheimer’s disease patients and healthy individuals in their abilities to clear Aβ from brain fluid and to protect neurons from Aβ toxicity. In the present study, we examined (1) the cytotoxicity of Alzheimer’s disease cerebrospinal fluid (CSF) compared to control CSF, (2) the ability of Alzheimer’s disease and control CSF to protect cells from Aβ toxicity and to promote cell-mediated clearance of Aβ and lastly (3) the effects of extracellular chaperones, normally found in CSF, on these processes. We show that the Alzheimer’s disease CSF samples tested were more toxic to cultured neuroblastoma cells than normal CSF. In addition, the Alzheimer’s disease CSF samples tested were less able to protect cells from Aβ-induced toxicity and less efficient at promoting macrophage-like cell uptake when compared to normal CSF. The addition of physiologically relevant concentrations of the extracellular chaperones, clusterin, haptoglobin and α₂-macroglobulin into CSF protected neuroblastoma cells from Αβ_1-42 toxicity and promoted Αβ_1-42 uptake in macrophage-like cells. These results suggest that extracellular chaperones are an important element of a system of extracellular protein folding quality control that protects against Aβ toxicity and accumulation.     

TOMM40 poly-T variants and cerebrospinal fluid amyloid beta levels in the elderly

A variable poly-T polymorphism in the TOMM40 gene, which is in linkage disequilibrium with APOE, was recently implicated with increased risk and earlier onset age for late-onset Alzheimer’s disease in APOE ε3 carriers. To elucidate potential neurobiological mechanisms underlying this association, we compared the effect of TOMM40 poly-T variants to the effect of APOE, an established LOAD-risk modulator, on cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau levels, in cognitively intact elderly subjects. APOE ε4 carriers showed significant reductions in Aβ 1-42 levels compared to non-ε4 carriers, but no differences were detected across TOMM40 variants. Neither Aβ 1-40 nor tau levels were affected by APOE or TOMM40.     

X-ray absorption and diffraction studies of the metal binding sites in amyloid <Emphasis Type="Italic">β</Emphasis>-peptide

A major source of neurodegeneration observed in Alzheimer’s disease is believed to be caused by the toxicity from reactive oxygen species produced in the brain mediated by the Aβ protein and mainly copper species. An atomic model of an amyloid β-peptide (Aβ) Cu²⁺ complex or at least the structure of the metal binding site is of great interest. Accurate information about the Cu-binding site of Aβ protein can facilitate simulation of redox chemistry using high level quantum mechanics. Complementary X-ray diffraction and X-ray absorption techniques can be employed to obtain such accurate information. This review provides a blend of X-ray diffraction results on amyloid structures and selected works on Aβ Cu²⁺ binding based on spectroscopic measurements with emphasis on the X-ray absorption technique. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Interaction between Aβ Peptide and α Synuclein: Molecular Mechanisms in Overlapping Pathology of Alzheimer’s and Parkinson’s in Dementia with Lewy Body Disease

Amyloidogenic proteins (Aβ peptide) in Alzheimer’s disease (AD) and alpha-synuclein (α-Syn) in Parkinson’s disease (PD) are typically soluble monomeric precursors, which undergo remarkable conformational changes and culminate in the form of aggregates in diseased condition. Overlap of clinical and neuropathological features of both AD and PD are observed in dementia with Lewy body (DLB) disease, the second most common form of dementia after AD. The identification of a 35-amino acid fragment of α-Syn in the amyloid plaques in DLB brain have raised the possibility that Aβ and α-Syn interact with each other. In this report, the molecular interaction of α-Syn with Aβ40 and/or Aβ42 are investigated using multidimensional NMR spectroscopy. NMR data in the membrane mimic environment indicate specific sites of interaction between membrane-bound α-Syn with Aβ peptide and vice versa. These Aβ–α-Syn interactions are demonstrated by reduced amide peak intensity or change in chemical shift of amide proton of the interacting proteins. Based on NMR results, the plausible molecular mechanism of overlapping pathocascade of AD and PD in DLB due to interactions between α-Syn and Aβ is described. To the best of our knowledge, it is the first report using multidimensional NMR spectroscopy that elucidates molecular interactions between Aβ and α-Syn which may lead to onset of DLB. An erratum to this article can be found at     

Novel Detox Gel Depot Sequesters β-Amyloid Peptides in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD), a debilitating neurodegenerative disease is caused by aggregation and accumulation of a 39–43 amino acid peptide (amyloid β or Aβ) in brain parenchyma and cerebrovasculature. The rational approach would be to use drugs that interfere with Aβ–Aβ interaction and disrupt polymerization. Peptide ligands capable of binding to the KLVFF (amino acids 16–20) region in the Aβ molecule have been investigated as possible drug candidates. Retro-inverso (RI) peptide of this pentapeptide, ffvlk, has been shown to bind artificial fibrils made from Aβ with moderate affinity. We hypothesized that a ‘detox gel’, which is synthesized by covalently linking a tetrameric version of RI peptide ffvlk to poly(ethylene glycol) polymer chains will act like a ‘sink’ to capture Aβ peptides from the surrounding environment. We previously demonstrated that this hypothesis works in an in vitro system. The present study extended this hypothesis to an in vivo mouse model of AD and determined the therapeutic effect of our detox gel. We injected detox gel subcutaneously to AD model mice and analyzed brain levels of Aβ-42 and improvement in memory parameters. The results showed a reduction of brain amyloid burden in detox gel treated mice. Memory parameters in the treated mice improved. No undesirable immune response was observed. The data strongly suggest that our detox gel can be used as an effective therapy to deplete brain Aβ levels. Considering recent abandonment of failed antibody based therapies, our detox gel appears to have the advantage of being a non-immune based therapy.     

Glutamate antibodies repress expression of <Emphasis Type="Italic">Dffb</Emphasis> gene in brain of rats in experimental Alzheimer’s disease

The rat model of Alzheimer’s disease including injection of neurotoxic fragment of β-amyloid protein Aβ_25–35 into giant-cell nuclei basalis of Meynert was used for experiments. We have investigated the influence of glutamate antibodies administered intranasally in a dose of 300 μg/kg after 1 h of the mentioned alteration on the level of expression of Dffb mRNA. Dffb gene codes caspase-dependent DNase, which participates in the internucleosomal fragmentation of genome DNA during apoptosis. On the third day after the injection of Aβ_25–35, we obtained a significant decrease in Dffb gene expression in the prefrontal cortex (37% decrease) and hippocampus (62% decrease) in the group of experimental animals compared to the control group. In the hypothalamus, there were no such differences. Seemingly, the repressing action of glutamate antibodies on the mRNA expression of the Dffb gene reflects the stabilization of processes that take place in the brain cells during experimental Alzheimer’s disease; meanwhile, the intensity of the apoptotic death of neurons and glial cells decreases.     

PS1 Expression is Downregulated by Gonadal Steroids in Adult Mouse Brain

Mutations in presenilin (PS) 1 and PS2 genes are associated with early onset (≤65 years) of Alzheimer’s disease (AD). PS1 is involved in γ-secretase mediated cleavage of β-amyloid precursor protein (APP), but its regulation is poorly understood. Sex steroids influence APP cleavage pathways resulting in reduced burden of both intra- and extra-cellular nonamyloidogenic products. As gonadal hormones are implicated in AD and their levels change with age, we have analyzed the effect of 17β-estradiol and testosterone on PS1 expression in the cerebral cortex of adult and old AKR mice of both sexes. Northern and Western-blot analysis revealed that PS1 mRNA and protein expression followed similar pattern of regulation. PS1 expression was downregulated by 17β-estradiol and testosterone in the cerebral cortex of females and adult male, but upregulated in old male mice. Such sex-dependent regulation of PS1 expression during aging by gonadal steroids might account for the PS-related brain functions.     

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade

Aβ vaccination as a therapeutic intervention of Alzheimer’s has many challenges, key among them is the regulation of inflammatory processes concomitant with excessive generation of free radicals seen during such interventions. Here we report the beneficial effects of melatonin on inflammation associated with Aβ vaccination in the central and peripheral nervous system of mice. Mice were divided into three groups (n = 8 in each): control, inflammation (IA), and melatonin-treated (IAM). The brain, liver, and spleen samples were collected after 5 days for quantitative assessment of plasma lipid peroxides (LPO), an oxidative stress marker, and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (Gpx). IA group mice have shown the elevated concentration of LPO significantly while there was a reduction at antioxidant enzyme levels. In addition, a significant (P < 0.05) reduction in neurotransmitters like dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) was also observed in the IA group mice. Nevertheless, their metabolites, such as homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P < 0.05) as compared to control. Samples were further evaluated at microscopic level to examine the neuropathological changes by immunohistochemical methods. Melatonin treatment effectively reversed these above changes and normalized the LPO and antioxidant enzyme levels (P < 0.05). Furthermore, melatonin salvaged the brain cells from inflammation. Our Immunohistochemical findings in the samples of melatonin-treated animals (IAM group) indicated diminished expression of glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (NfκB) than those observed in the IA group samples. Our results suggest that administration of melatonin protects inflammation associated with Aβ vaccination, through its direct and indirect actions and it can be an effective adjuvant in the development of vaccination in immunotherapy for Alzheimer’s disease (AD).     

Relation of Plasma Homocysteine to Plasma Amyloid Beta Levels

Background Elevated plasma homocysteine and amyloid β (Aβ) have been associated with Alzheimer’s disease (AD). We investigated the cross-sectional association between these biomarkers. Methods We used linear regression to relate plasma homocysteine and Aβ adjusting for age, gender, creatinine, APOE-ε4, and ethnic group in 327 persons aged 78 ± 6.6 years. Results Plasma homocysteine correlated with age, serum creatinine, plasma Aβ40 and Aβ42, and was inversely correlated with serum vitamin B12, and folate. Aβ42, but not Aβ40, was related to later development of dementia. Homocysteine was related to higher Aβ40 levels (coefficient = 2.0; P < 0.0001) and this association was attenuated after adjustment for creatinine (coefficient = 1.0; P < 0.0001). The crude association between homocysteine and Aβ42 was weaker (coefficient = 0.5; P = 0.01) and became non-significant after adjustment for creatinine (coefficient = 0.4; P = 0.06). These associations were unrelated to ethnicity, the presence of APOE-ε4 or dementia. Analyses by quartiles of homocysteine showed that these association were driven primarily by the fourth quartile. Conclusions Plasma homocysteine is directly related to Aβ40. The association with Aβ42 is not significant. These results seem to indicate that homocysteine is related to aging but not specifically to AD. Special issue dedicated to John P. Blass.     

Elevation in Plasma Abeta42 in Geriatric Depression: A Pilot Study

Elevated plasma amyloid beta 1–42 (Aβ42) level has been linked to increased risk for incident AD in cognitively-intact elderly. However, plasma Aβ levels in individuals with late-life depression (LLMD), especially those with a late age of onset of first depressive episode, who are at a particularly increased risk for Alzheimer’s disease, have not been studied. We compared plasma Aβ in 47 elderly with LLMD with 35 controls and examined its relationships to age of onset of first depressive episode, antidepressant treatment (paroxetine or nortriptyline), and indices of platelet activation (platelet factor 4 and beta-thromboglobulin) and brain abnormalities. Results indicated that plasma Aβ42 levels and the Aβ42/40 ratio were elevated in the LLMD group relative to controls in the overall group analyses and in the age- and gender-matched groups. MRI data indicated that higher Aβ42/40 ratio was associated with greater severity of total white matter hyperintensity burden in LLMD. Plasma Aβ levels in LLMD were not influenced by age of onset of first depressive episode or antidepressant treatment and were not related to indices of platelet activation. Our preliminary results suggest that increased plasma Aβ42 and Aβ42/40 ratio are present in geriatric depression, and future studies should be done to confirm these findings and to determine their relationship to cognitive decline and brain abnormalities associated with LLMD.Presented at the 43rd Annual Meeting, American College of Neuropsychopharmacology, San Juan, Puerto Rico, December 12–16, 2004.     

Mitochondria and Alzheimer’s disease: amyloid-β peptide uptake and degradation by the presequence protease,hPreP

Several lines of evidence suggest mitochondrial dysfunction as a possible underlying mechanism of Alzheimer’s disease (AD). Accumulation of the amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathogenesis of AD, has been detected in brain mitochondria of AD patients and AD transgenic mouse models. In vitro evidence suggests that the Aβ causes mitochondrial dysfunction e.g. oxidative stress, mitochondrial fragmentation and decreased activity of cytochrome c oxidase and TCA cycle enzymes. Here we review the link between mitochondrial dysfunctions and AD. In particular we focus on the mechanism for Aβ uptake by mitochondria and on the recently identified Aβ degrading protease in human brain mitochondria.     

Interactions of Npc1 and amyloid accumulation/deposition in the APP/PS1 mouse model of Alzheimer’s

Although Niemann-Pick C1 disease has frequently been called “juvenile Alzheimer’s”, the effects of introducing Npc1 mutations into a mouse model of Alzheimer’s have not previously been performed. We have crossed Npc1 ^+/− mice with APP/PS1 “Alzheimer’s” mice and studied Aβ42 accumulation and amyloid plaque formation. Mice heterozygous for Npc1 and positive for the APP and PS1 transgenes accumulated Aβ42 more rapidly than the APP/PS1 controls and this correlated, as expected, with the area of amyloid plaques. We conclude that the alterations of intracellular cholesterol present in Npc1 ^+/− mice can influence the progress of Alzheimer’s disease in the APP/PS1 mouse model.     

Cytoplasmic tail adaptors of alzheimer’s amyloid-β protein precursor

Alzheimer’s disease is characterized pathologically by senile plaques in the brain. The major component of senile plaques is amyloid-β (Aβ), which is cleaved from Alzheimer’s Aβ protein precursor (AβPP). Recently, information regarding the cytoplasmic tail of AβPP has started to emerge, opening up various insights into the physiological roles of AβPP and its pathological role in Alzheimer’s disease. The cytoplasmic domain of AβPP shares the evolutionarily conserved GYENPTY motif, which binds to a number of adaptor proteins containing the phosphotyrosine interaction domain (PID). Among the PID-containing proteins, this article focuses on four groups of adaptor proteins of AβPP: Fe65, X11, mDab1, and c-Jun N-terminal kinase-interacting protein 1b/islet-brain 1. These two authors contributed equally to this study.     

Three-dimensional colocalization analysis of plasma-derived apolipoprotein B with amyloid plaques in APP/PS1 transgenic mice

Parenchymal accumulation of amyloid-beta (Aβ) is a hallmark pathological feature of Alzheimer’s disease. An emerging hypothesis is that blood-to-brain delivery of Aβ may increase with compromised blood–brain barrier integrity. In plasma, substantial Aβ is associated with triglyceride-rich lipoproteins (TRLs) secreted by the liver and intestine. Utilizing apolipoprotein B as an exclusive marker of hepatic and intestinal TRLs, here we show utilizing an highly sensitive 3-dimensional immuno-microscopy imaging technique, that in APP/PS1 amyloid transgenic mice, concomitant with substantially increased plasma Aβ, there is a significant colocalization of apolipoprotein B with cerebral amyloid plaque. The findings are consistent with the possibility that circulating lipoprotein-Aβ contributes to cerebral amyloidosis.     

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and β-amyloid

Summary Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer’s disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE₁-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA₁ was more effective than PGE₁ in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and β-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and β-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and β-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and β-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.     

In vivo effects of APP are not exacerbated by BACE2 co-overexpression: behavioural characterization of a double transgenic mouse model

Down syndrome, the most common genetic disorder leading to mental retardation, is caused by the presence of all or part of an extra copy of chromosome 21. At relatively early ages, Down syndrome patients develop progressive formation and extracellular aggregation of amyloid-β peptide, considered as one of the causal factors for the pathogenesis of Alzheimer’s disease. This neuropathological hallmark has been attributed to the overexpression of APP but could also be contributed by other HSA21 genes. BACE2 maps to HSA21 and is homologous to BACE1, a β-secretase involved in the amyloidogenic pathway of APP proteolysis, and thus it has been hypothesized that the co-overexpression of both genes could contribute to Alzheimer’s like neuropathology present in Down syndrome. The aim of the present study has been to analyse the impact of the co-overexpression of BACE2 and APP, using a double transgenic mouse model. Double transgenic mice did not present any neurological or sensorimotor alterations, nor genotype-dependent anxiety-like behaviour or age-associated cognitive dysfunction. Interestingly, TgBACE2-APP mice showed deregulation of BACE2 expression levels that were significantly increased with respect to single TgBACE2 mice. Co-overexpression of BACE2 and APP did not increase amyloid-β peptide concentration in brain. Our results suggest that the in vivo effects of APP are not exacerbated by BACE2 co-overexpression but may have some protective effects in specific behavioural and cognitive domains in transgenic mice.     

Neprilysin Deficiency-Dependent Impairment of Cognitive Functions in a Mouse Model of Amyloidosis

Alzheimer’s disease, responsible for the vast majority of dementia cases in the elderly population, is caused by accumulation of toxic levels of amyloid β peptide (Aβ) in the brain. Neprilysin is a major enzyme responsible for the degradation of Aβ in vivo. We have previously shown that elevation of neprilysin levels in the brain delays the deposition of Aβ -plaques in a mouse model of amyloidosis and that lack of neprilysin leads to increased Aβ generation and to signs of incipient neurodegeneration in mouse brains. This study was designed to test whether low brain levels of neprilysin affect the amyloid pathology or perturb the learning and memory performance of mice. Double-mutated mice carrying a targeted depletion of one allele of Mme, the gene encoding neprilysin, and over-expressing human amyloid precursor protein (APP), exhibited a reinforced amyloid pathology in comparison with their APP transgenic littermates. Moreover, in contrast to their parental lines, these mice were impaired in the Morris water maze learning and memory paradigm and showed facilitated extinction in the conditioned taste aversion test. These data suggest that even a partial neprilysin deficiency, as is found during aging, exacerbates amyloid pathology and may impair cognitive functions. Special issue to Honor Dr. Akitane Mori.     

Dopamine β-hydroxylase: two polymorphisms in linkage disequilibrium at the structural gene DBH associate with biochemical phenotypic variation

Levels of the enzyme dopamine β-hydroxylase (DβH) in the plasma and cerebrospinal fluid (CSF) are closely related biochemical phenotypes. Both are under strong genetic control. Linkage and association studies suggest the structural gene encoding DβH (locus name, DΒH) is a major locus influencing plasma activity of DβH. This study examined relationships of DBH genotype determined at two polymorphic sites (a previously described GT repeat, referred to as the DBH STR and a single-base substitution at the 3’ end of DBH exon 2, named DBH*444 g/a), to CSF levels of DβH protein in European-American schizophrenic patients, and to plasma DβH activity in European-American patients with mood or anxiety disorders. We also investigated linkage disequilibrium (LD) between the polymorphisms in the pooled samples from those European-American subjects (n=104). Alleles of DBH*444 g/a were associated with differences in mean values of CSF DβH levels. Alleles at both polymorphisms were associated with plasma DβH activity. Significant LD was observed between respective alleles with similar apparent influence on biochemical phenotype. Thus, allele A3 of the DBH STR was in positive LD with DBH*444a, and both alleles were associated with lower plasma DβH activity. DBH STR allele A4 was in positive LD with DBH*444 g, and both alleles were associated with higher plasma DβH activity. The results confirm that DBH is a major quantitative trait locus for plasma DβH activity, and provide the first direct evidence that DBH also influences CSF DβH levels. Both polymorphisms examined in this study appear to be in LD with one or more functional polymorphisms that mediate the influence of allelic variation at DBH on DβH biochemical phenotypic variation Received: 14 October 1997 / Accepted: 31 December 1997     

CSF total tau, Aβ42 and phosphorylated tau protein as biomarkers for Alzheimer’s disease

With the arrival of effective symptomatic treatments and the promise of drugs that may delay progression, we now need to identify Alzheimer’s disease (AD) at an early stage of the disease. To diagnose AD earlier and more accurately, attention has been directed toward peripheral biochemical markers. This article reviews promising potential cerebrospinal fluid (CSF) biomarkers for AD focussing on their role in clinical diagnosis. In particular, two biochemical markers, CSF total tau (t-tau) protein and the 42 amino acid form of β-amyloid (Aβ42), perform satisfactorily enough to achieve a role in the clinical diagnostic settings of patients with dementia together with the cumulative information from basic clinical work-up, genetic screening, and brain imaging. These CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. In order to discriminate AD from other primary dementia disorders, however, more accurate and specific markers are needed. Preliminary evidence strongly suggests that quantification of tau phosphorylated at specific sites in CSF improves early detection, differential diagnosis, and tracking of disease progression in AD.     

Systemic Oxidative Stress Associated with the Neurological Diseases of Aging

Markers of oxidative stress were measured in blood samples of 338 subjects (965 observations): Alzheimer’s, vascular dementia, diabetes (type II) superimposed to dementias, Parkinson’s disease and controls. Patients showed increased thiobarbituric acid reactive substances (+21%; P < 0.05), copper-zinc superoxide dismutase (+64%; P < 0.001) and decreased antioxidant capacity (−28%; P < 0.001); pairs of variables resulted linearly related across groups (P < 0.001). Catalase and glutathione peroxidase, involved in discrimination between diseases, resulted non-significant. When diabetes is superimposed with dementias, changes resulted less marked but significant. Also, superoxide dismutase resulted not linearly correlated with any other variable or age-related (pure Alzheimer’s peaks at 70 years, P < 0.001). Systemic oxidative stress was significantly associated (P ≪ 0.001) with all diseases indicating a disbalance in peripheral/adaptive responses to oxidative disorders through different free radical metabolic pathways. While other changes—methionine cycle, insulin correlation—are also associated with dementias, the responses presented here show a simple linear relation between prooxidants and antioxidant defenses.