Decreased brain creatine levels in elderly apolipoprotein E ε4 carriers

Summary. Apolipoprotein E (ApoE) genotype has been shown to influence results in neuroimaging studies using a number of various imaging modalities. No in vivo data exists on whether or not there are ApoE-related changes observable by proton magnetic resonance spectroscopy (MRS). In this study we measured absolute peak areas of proton MR spectra obtained from the occipital cortex in 22 non-demented elderly with (n = 8) or without (n = 14) the ApoE ε4 allele. No statistically significant differences were found in levels of N-acetyl aspartate, myo-inositol, or choline containing compounds between the groups. Instead, compared with the non-carriers, the levels of creatine were significantly lower in the ε4 carriers, suggesting increased metabolic demands in the brain of the ε4 carriers. The levels of creatine also correlated significantly with age and performance on the Mini-Mental State Examination test in the ε4 carriers, but not in the non-carriers. These findings may be of significant clinical interest as potential indicator of incipient AD, and also from therapeutical point of view given the potential neuroprotective effects of creatine. Received February 18, 2002; accepted August 5, 2002 Published online December 9, 2002 Acknowledgements This study was supported by the Research Council for Health of the Academy of Finland, the Finnish Neurology Foundation, the Instrumentarium Research Foundation, and the Farmos Research Foundation. Authors' address: M. Laakso, Department of Neurology, Bldg. 5, Kuopio University Hospital, P.O.Box 1777, 70211 Kuopio, Finland, e-mail: mikko.laakso@uku.fi Abbreviations AD Alzheimer's disease, ApoE apolipoprotein E, MI myo-inositol, MMSE Mini-Mental State Examination, MRI magnetic resonance imaging, MRS magnetic resonance spectroscopy, NAA N-acetyl aspartate.     

Changes in brain β-amyloid deposition and aquaporin 4 levels in response to altered agrin expression in mice

Conditions that compromise the blood-brain barrier (BBB) have been increasingly implicated in the pathogenesis of Alzheimer disease (AD). AGRIN is a heparan sulfate proteoglycan found abundantly in basement membranes of the cerebral vasculature, where it has been proposed to serve a functional role in the BBB. Furthermore, AGRIN is the major heparan sulfate proteoglycan associated with amyloid plaques in AD brains. To examine the relationship of AGRIN, the BBB, and AD-related pathologies, we generated mice in which the Agrn gene was deleted from either endothelial cells or neurons using gene targeting or was overexpressed using a genomic transgene construct. These mice were combined with a transgenic model of AD that over expresses disease-associated forms of amyloid precursor protein and presenilin 1. In mice lacking endothelial cell expression of Agrn, the BBB remained intact but aquaporin 4 levels were reduced, indicating that the loss of AGRIN affects BBB-associated components. This change in Agrn resulted in an increase in β-amyloid (Aβ) in the brain. Conversely, overexpression of Agrn decreased Aβ deposition, whereas elimination of Agrn from neurons did not change Aβ levels. These results indicate that AGRIN is important for maintaining BBB composition and that changes in Agrn expression (particularly vessel-associated AGRIN) influence Aβ homeostasis in mouse models of AD.     

Presence of apolipoprotein E on extracellular neurofibrillary tangles and on meningeal blood vessels precedes the Alzheimer β-amyloid deposition

The localization of apolipoprotein E (ApoE) has been examined immunohistochemically in the autopsied brains of middle-aged and old-aged control subjects, with and without amyloid protein (A) deposits, and of Alzheimer's disease patients. Senile plaques were consistently labeled with ApoE antiserum even in the very early stage of senile plaque formation seen in the fifth decade. In the cerebellar molecular layer, small dots of ApoE immunoreactivity, which were prominent in the Alzheimer's disease subjects, were observed in addition to immunoreactivity in diffuse plaques. ApoE antisera labeled all of the extracellular neurofibrillary tangles (NFT), whereas only a small minority of extracellular NFT were positive for A. A punctate pattern of ApoE immunoreactivity was seen at the media of the meningeal vessels lacking amyloid, when senile plaques were present in the nearby cortex. In the early stage of amyloid angiopathy, the distribution of ApoE immunoreactivity was much more extensive than that of A positivity. These findings suggest that ApoE accumulates in the early stage of senile plaque formation and, furthermore, that ApoE accumulation precedes A deposition in extracellular NFT and amyloid angiopathy.     

Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasma from Alzheimer’s disease patients and controls

The apolipoprotein E (ApoE) ε4 allele is the strongest risk factor of sporadic Alzheimer’s disease (AD), however, the fluid concentrations of ApoE and its different isoforms (ApoE2, ApoE3 and ApoE4) in AD patients and among APOE genotypes (APOE ε2, ε3, ε4) remain controversial. Using a novel mass spectrometry-based method, we quantified total ApoE and specific ApoE isoform concentrations and potential associations with age, cognitive status, cholesterol levels and established AD biomarkers in cerebrospinal fluid (CSF) from AD patients versus non-AD individuals with different APOE genotypes. We also investigated plasma total ApoE and ApoE isoform composition in a subset of these individuals. In total n = 43 AD and n = 43 non-AD subjects were included. We found that CSF and plasma total ApoE levels did not correlate with age or cognitive status and did not differ between AD and non-AD subjects deeming ApoE as an unfit diagnostic marker for AD. Also, whereas CSF ApoE levels did not vary between APOE genotypes APOE ε4 carriers exhibited significantly decreased plasma ApoE levels attributed to a specific decrease in the ApoE4 isoform concentrations. CSF total ApoE concentrations were positively associated with CSF, total tau, tau phosphorylated at Thr181 and Aβ1-42 of which the latter association was weaker and only present in APOE ε4 carriers indicating a differential involvement of ApoE in tau versus Aβ-linked neuropathological processes. Future studies need to elucidate whether the observed plasma ApoE4 deficiency is a life-long condition in APOE ?4 carriers and whether this decrease in plasma ApoE predisposes APOE ?4 carriers to AD.     

Oxidative stress in the production and expression of neurotoxic β-amyloid

Although there is not yet any in vivo evidence of the neurotoxic action of β-amyloid in humans, it is well established that the insoluble form of full length β-amyloid 1-40, and the fragment comprised of amino acids 25-35, are both toxic in vitro to neurons in tissue culture. β-amyloid 25-35 increases cytosolic calcium in rat PC12 cells and in rat cortical neurons in primary culture by facilitating the entry of extracellular calcium into the cell. This effect is not altered by calcium channel blocking drugs but is prevented by U-83836E, one of the lazaroid anti-oxidant drugs, and by vitamin E. Similarly, the neurotoxic actions of β-amyloid 25-35 are also prevented by U-83836E and by vitamin E. These observations indicate that the actions of β-amyloid 25-35 are mediated by free radicals. In vivo, β-amyloid 1-40 is cleaved from a precursor protein that appears to be synthesized and inserted into cellular membranes following damage to cells. To form neurotoxic β -amyloid, the precursor protein must be cleaved within the transmembrane portion of its structure. In spite of extensive world-wide effort, an enzyme capable of doing this has not been found. However, a peroxidation cascade propagated through the lipid bilayer of the cellular membrane would cleave the precursor protein at a site needed to form β-amyloid. If this is the case, then free radicals would play a role both in the formation of β-amyloid and in its neurotoxic actions.     

Physiological levels of β-amyloid induce cerebral vessel dysfunction and reduce endothelial nitric oxide production

Abstract

β-amyloid (Aβ), the major component of senile plaques in Alzheimer's disease (AD), normally circulates in the blood at nanomolar levels but is elevated in AD. Previous studies have found that high concentrations (10^-5 -10^-4 M) of Aβ result in neuronal cell death. Here we show that physiological levels of soluble Aβ can induce dysfunction in perfused rat cerebral vessels and in cultured endothelial cells. At concentrations of 10^-9 -10^-6 M, Aβ induced a significant concentration-dependent reduction of NO production in endothelial cells. At 10^-8 M, Aβ significantly decreased the sensitivity of cerebral vessels to acetylcholine (ACh), an endothelium dependent vasodilator. At 10^-7 M and higher concentrations, Aβ significantly reduced the maximum response of vessels to ACh, and induced significant endothelial cell death. Aβ (10^-9 -10^-5 M) did not cause any detectable change in nitric oxide synthase levels. The results suggest that a modest increase in the concentration of Aβ above its normal physiological level in the circulation, as found in the early stages of AD, results in decreased NO production and vessel sensitivity to endothelium-dependent vasodilation that could lead to constricted blood vessels and ischemia in the surrounding tissue. Further increases in Aβ concentration, which may occur in the later stages of AD, result in cell death and decreased maximum vasodilator response of cerebral vessels. [Neurol Res 2001; 23: 506-512]     

Genes and mechanisms involved in β-amyloid generation and Alzheimer’s disease

Alzheimer's disease is characterized by the invariable accumulation of senile plaques that are predominantly composed of amyloid beta-peptide (Abeta). Abeta is generated by proteolytic processing of the beta-amyloid precursor protein (betaAPP) involving the combined action of beta- and gamma-secretase. Cleavage within the Abeta domain by alpha-secretase prevents Abeta generation. In some very rare cases of familial AD (FAD), mutations have been identified within the betaAPP gene. These mutations are located close to or at the cleavage sites of the secretases and pathologically effect betaAPP processing by increasing Abeta production, specifically its highly amyloidogenic 42 amino acid variant (Abeta42). Most of the mutations associated with FAD have been identified in the two presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the betaAPP gene, mutations in PS1 and PS2 cause the increased generation of Abeta42. PS1 has been shown to be functionally involved in Notch signaling, a key process in cellular differentation, and in betaAPP processing. A gene knock out of PS1 in mice leads to an embryonic lethal phenotype similar to that of mice lacking Notch. In addition, absence of PS1 results in reduced gamma-secretase cleavage and leads to an accumulation of betaAPP C-terminal fragments and decreased amounts of Abeta. Recent work may suggest that PS1 could be the gamma-secretase itself, exhibiting the properties of a novel aspartyl protease. Mutagenesis of either of two highly conserved intramembraneous aspartate residues of PS1 leads to reduced Abeta production as observed in the PS1 knockout. A corresponding mutation in PS2 interfered with betaAPP processing and Notch signaling suggesting a functional redundancy of both presenilins. In this issue, some of the recent work on the molecular mechanisms involved in Alzheimer's disease (AD) as well as novel diagnostic approaches and risk factors for AD will be discussed. In the first article, we like to give an overview on mechanisms involved in the proteolytic generation of Amyloid beta-peptide (Abeta), the major pathological player of this devastating disease. In the second part of this article recent results will be described, which demonstrate an unexpected biological and pathological function of an AD associated gene.     

Cerebral β-amyloid deposition is augmented by the –491AA promoter polymorphism in non-demented elderly individuals bearing the apolipoprotein E ε4 allele

The apolipoprotein E epsilon4 allele (APOE, gene; apoE, protein) is widely accepted as a risk factor for Alzheimer's disease (AD). Our previous studies found that APOEepsilon4 promotes AD pathogenesis by fostering the early deposition of the amyloidogenic peptide Abeta in the aging brain. Recent reports suggest that polymorphisms in the upstream promoter region of APOE differentially affect the production of apoE and also may have an important influence on the probability of developing AD. In this study, we asked whether APOE promoter -491 (A/T) variants interact with APOE polymorphisms to modulate the degree of beta-amyloid- and tau-related pathology in the medial temporal lobe of the non-demented elderly. Our results confirm that APOEepsilon4 is associated with increased formation of senile plaques, cerebrovascular amyloid, and neurofibrillary tangles in the medial temporal lobe. We also found that homozygosity for A at position -491 of the APOE promoter (-491AA) correlates with increased Abeta17-24 and Abeta42 deposition in APOEepsilon4-positive cases, but not in cases lacking the epsilon4 allele. In comparison, Abeta burden is significantly less in epsilon4 carriers with the -491AT and -491TT promoter allelotypes. There was no effect of -491 polymorphisms on Abeta40 deposition (which is relatively sparse in the non-demented elderly), on the number of activated microglia, or on the amount of neurofibrillary tangles. We conclude that the amyloidogenic effects of apoE4 are exacerbated by polymorphisms in the APOE promoter that enhance apoE production.     

Intraneuronal β-amyloid accumulation and synapse pathology in Alzheimer’s disease

The aberrant accumulation of aggregated β-amyloid peptides (Aβ) as plaques is a hallmark of Alzheimer’s disease (AD) neuropathology and reduction of Aβ has become a leading direction of emerging experimental therapies for the disease. The mechanism(s) whereby Aβ is involved in the pathophysiology of the disease remain(s) poorly understood. Initially fibrils, and subsequently oligomers of extracellular Aβ have been viewed as the most important pathogenic form of Aβ in AD. More recently, the intraneuronal accumulation of Aβ has been described in the brain, although technical considerations and its relevance in AD have made this a controversial topic. Here, we review the emerging evidence linking intraneuronal Aβ accumulation to the development of synaptic pathology and plaques in AD, and discuss the implications of intraneuronal β-amyloid for AD pathology, biology, diagnosis and therapy.     

Monitoring pathological assembly of tau and β-amyloid proteins in Alzheimer's disease

This double-labelling confocal microscopy study of the neuropathology of Alzheimer's disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects -amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse -amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tanglebearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for -pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.Supported in part by CONACyT grant #1624-N9208 (to R.M.), the Medical Research Council (U.K.), Zeneca Pharmaceuticals and the Alzheimer Disease Research Fund and the Leopold Muller Estate     

Does simvastatin affect mood and steroid hormone levels in hypercholesterolemic men? A randomized double-blind trial

Epidemiological and clinical studies have suggested that powerful cholesterol lowering may have adverse effects on mood and psychological well-being. Inhibition of cholesterol biosynthesis by simvastatin (a hydroxymethyl glutaryl coenzyme A reductase inhibitor) may also reduce steroid hormone biosynthesis. To explore if mood changes are related with steroid hormone levels, we designed a randomized double-blind placebo-controlled crossover trial. The separate and combined effects of a Mediterranean-type diet intervention and treatment with simvastatin 20 mg/day PO for 12 weeks were studied in 120 hypercholesterolemic but otherwise healthy middle-aged men. Psychological functioning was assessed with questionnaires, and steroid hormone levels in blood were assayed radioimmunologically before and after the treatments. Simvastatin resulted in a statistically significant increase of depression and somatization without changes in the anxiety, hostility or aggression scores. Mood changes seemed to be unrelated with the statistically significant but clinically insignificant decline in serum testosterone levels and unrelated with the increase in serum dehydroepiandrosterone levels.     

Impaired β-amyloid secretion in Alzheimer's disease pathogenesis

A central question in Alzheimer's disease (AD) research is what role β-amyloid peptide (Aβ) plays in synaptic dysfunction. Synaptic activity increases Aβ secretion, potentially inhibiting synapses, but also decreases intraneuronal Aβ, protecting synapses. We now show that levels of secreted Aβ fall with time in culture in neurons of AD-transgenic mice, but not wild-type mice. Moreover, the ability of synaptic activity to elevate secreted Aβ and reduce intraneuronal Aβ becomes impaired in AD-transgenic but not wild-type neurons with time in culture. We demonstrate that synaptic activity promotes an increase in the Aβ-degrading protease neprilysin at the cell surface and a concomitant increase in colocalization with Aβ42. Remarkably, AD-transgenic but not wild-type neurons show reduced levels of neprilysin with time in culture. This impaired ability to secrete Aβ and reduce intraneuronal Aβ has important implications for the pathogenesis and treatment of AD.     

Neocortical β-amyloid area is associated with dementia and APOE in the oldest-old

ObjectiveApolipoprotein E (APOE) ε2 carriers may be protected from dementia because of reduced levels of cortical β-amyloid. In the oldest-old, however, APOE ε2 carriers have high β-amyloid plaque scores and preserved cognition. We compared different measures of β-amyloid pathology across APOE genotypes in the oldest-old, and their relationship with dementia.MethodsThe study included 96 participants from The 90+ Study. Using all information, dementia diagnoses were made. Neuropathological examination included staging for amyloid plaques and β-amyloid cortical percent area stained by NAB228 antibody.ResultsBoth APOE ε2 and APOE ε4 carriers had high Consortium to Establish a Registry for Alzheimer’s Disease plaque scores. However, APOE ε2 carriers had low cortical β-amyloid percent areas. β-amyloid percent area was associated with dementia across APOE genotypes.ConclusionsLower levels of percent area in APOE ε2 carriers may reflect lower total β-amyloid and may contribute to APOE ε2 carriers’ decreased risk of dementia, despite high β-amyloid plaque scores. The relationship between β-amyloid plaques and dementia in the oldest-old may vary by APOE genotype.     

CSF levels of tau, β-amyloid1–42 and GAP-43 in frontotemporal dementia, other types of dementia and normal aging

Summary. Cerebrospinal fluid (CSF) levels of tau, β-amyloid_1–42 and growth-associated protein 43 (GAP-43) were studied in patients with frontotemporal dementia (FTD; n = 17), Alzheimer's disease (AD; n = 60), subcortical white-matter dementia (SWD; n = 24), Parkinson's disease (PD; n = 23) and dysthymia (n = 19) and in age-matched controls (n = 32). CSF-tau was significantly increased only in AD, and CSF-β-amyloid_1–42 was significantly decreased in AD and SWD as compared to controls, and in AD compared to FTD. CSF-GAP-43 was significantly decreased only in PD. The GAP-43/tau ratio was decreased in all the patient groups except the dysthymia group compared to controls. A positive correlation was found between CSF-GAP-43 and CSF-tau in all groups. The results suggest normal levels of CSF-tau and CSF-β-amyloid_1–42 in FTD, which will aid in the clinical separation of FTD from AD. In SWD, decreased levels of CSF-β-amyloid_1–42 suggest concomitant involvement of vascular and amyloid protein mechanisms. Received July 15, 1999; accepted October 1, 1999     

Does apolipoprotein E influence learning and memory in the nondemented oldest old?

The objective of this study was to analyze the relationship of the apolipoprotein E (apoE) epsilon4 and epsilon2 alleles to learning and memory performances in the nondemented oldest old. Forty-six nondemented persons aged 85 years or over from a randomly selected group of 128 subjects in Vantaa, Finland, were studied. ApoE genotyping was performed using the minisequencing technique. A structured clinical examination and interview were carried out. The test variables studied were learning and memory scores (from the Fuld Object-Memory Evaluation), verbal fluency, and conceptualization (the Similarities subtest of the WAIS-R). We compared apoE-epsilon4 carriers to noncarriers and apoE-epsilon2 carriers to noncarriers. No statistically significant differences were found in any of the test variables. The results failed to confirm the hypotheses that poor cognitive performance is associated with the apoE-epsilon4 allele and good performance with the apoE-epsilon2 allele in the oldest old. This suggests that the apoE alleles do not have a detectable relationship to learning and memory in nondemented very elderly people.