Dysregulation of the renin-angiotensin system in vascular dementia.

The renin-angiotensin system (RAS) regulates systemic and cerebral blood flow and is dysregulated in dementia. The major aim of this study was to determine if RAS signalling is dysregulated in vascular dementia. We measured markers of RAS signalling in white matter underlying the frontal and occipital cortex in neuropathologically confirmed cases of vascular dementia (n = 42), Alzheimer's disease (n = 50), mixed AD/VaD (n = 50) and age-matched controls (n = 50). All cases were stratified according to small vessel disease (SVD) severity across both regions. ACE-1 and ACE-2 protein and activity was measured by ELISA and fluorogenic peptide assays respectively, and angiotensin peptide (Ang-II, Ang-III and Ang-(1-7)) levels were measured by ELISA. ACE-1 protein level and enzyme activity, and Ang-II and Ang-III, were elevated in the white matter in vascular dementia in relation to SVD severity. ACE-1 and Ang-II protein levels were inversely related to MAG:PLP1 ratio, a biochemical marker of brain tissue oxygenation that when reduced indicates cerebral hypoperfusion, in a subset of cases. ACE-2 level was elevated in frontal white matter in vascular dementia. Ang-(1-7) level was elevated across all dementia groups compared to age-matched controls but was not related to SVD severity. RAS signalling was not altered in the white matter in Alzheimer's disease. In the overlying frontal cortex, ACE-1 protein was reduced and ACE-2 protein increased in vascular dementia, whereas angiotensin peptide levels were unchanged. These data indicate that RAS signalling is dysregulated in the white matter in vascular dementia and may contribute to the pathogenesis of small vessel disease.

Regional differences in effects of APOE ε4 on cognitive impairment in non-demented subjects.

BACKGROUND: The APOE ε4 allele is a risk factor for Alzheimer's disease (AD). APOE ε4 is common in non-demented subjects with cognitive impairment. In both healthy people and people with AD, its prevalence has a north-south gradient across Europe. In the present study, we investigated whether the relation between the APOE ε4 allele and cognitive impairment varied across Northern, Middle and Southern Europe. We also investigated whether a north-south gradient existed in subjects with subjective cognitive impairment (SCI), amnestic mild cognitive impairment (MCI) and non-amnestic MCI. METHODS: Data from 16 centers across Europe were analyzed. RESULTS: A north-south gradient in APOE ε4 prevalence existed in the total sample (62.7% for APOE ε4 carriers in the northern region, 42.1% in the middle region, and 31.5% in the southern region) and in subjects with SCI and amnestic MCI separately. Only in Middle Europe was the APOE ε4 allele significantly associated with poor performance on tests of delayed recall and learning, as well as with the amnestic subtype of MCI. CONCLUSION: The APOE ε4 allele frequencies in subjects with SCI and amnestic MCI have a north-south gradient. The relation between the APOE ε4 allele and cognition is region dependent.

Endothelin-converting enzyme-1 in Alzheimer's disease and vascular dementia.

AIMS: Alzheimer's disease (AD) is believed to be caused by the accumulation of amyloid beta (Aß) peptide within the brain. Endothelin-converting enzyme-1 and 2 (ECE-1 and ECE-2) are expressed in endothelial cells and neurones, respectively, and both cleave 'big endothelin' to produce the vasoconstrictor endothelin-1 (ET-1). ECE-1 and ECE-2 also degrade Aß. AD patients have regionally reduced microvascular blood flow in the brain, with impaired endothelium-dependent relaxation and cerebrovascular autoregulation, and abnormal production of ET-1 has been demonstrated in mice overexpressing amyloid precursor protein. We recently found ECE-2 mRNA and protein to be elevated in the brain in AD. In vitro, expression of ECE-2 was upregulated by Aß. Our aims for this study were to examine expression of ECE-1 (which has 57% homology with ECE-2) in temporal cortex from patients with AD, vascular dementia (VaD) and controls. METHODS: We examined the distribution of ECE-1 with immunohistochemistry, and measured ECE-1 mRNA by real-time polymerase chain reaction (PCR). ECE-1 protein levels were measured by western blot, and results analysed before and after adjustment for factor VIII-related antigen. RESULTS: We showed ECE-1 to be in vascular endothelial cells. We did not find significant differences in ECE-1 mRNA or protein levels (either full-length ECE-1 or the soluble spliced variant, ECE-1sv) in AD or VaD compared with controls. CONCLUSIONS: Our findings suggest that any disease-specific contribution of ECE-1 to the accumulation of Aß or reduction in local microvascular blood flow in AD or VaD is probably small, with abnormal production of ET-1 being more likely to reflect Aß-mediated upregulation of ECE-2.

BCAT-induced autophagy regulates Aß load through an interdependence of redox state and PKC phosphorylation-implications in Alzheimer's disease.

Leucine, nutrient signal and substrate for the branched chain aminotransferase (BCAT) activates the mechanistic target of rapamycin (mTORC1) and regulates autophagic flux, mechanisms implicated in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease (AD). BCAT is upregulated in AD, where a moonlighting role, imparted through its redox-active CXXC motif, has been suggested. Here we demonstrate that the redox state of BCAT signals differential phosphorylation by protein kinase C (PKC) regulating the trafficking of cellular pools of BCAT. We show inter-dependence of BCAT expression and proteins associated with the P13K/Akt/mTORC1 and autophagy signalling pathways. In response to insulin or an increase in ROS, BCATc is trafficked to the membrane and docks via palmitoylation, which is associated with BCATc-induced autophagy through PKC phosphorylation. In response to increased levels of BCATc, as observed in AD, amyloid ß (Aß) levels accumulate due to a shift in autophagic flux. This effect was diminished when incubated with leucine, indicating that dietary levels of amino acids show promise in regulating Aß load. Together these findings show that increased BCATc expression, reported in human AD brain, will affect autophagy and Aß load through the interdependence of its redox-regulated phosphorylation offering a novel target to address AD pathology.

Susceptibility locus for Alzheimer's disease on chromosome 10.

The apolipoprotein E (APOE) gene is the only genetic risk factor that has so far been linked to risk for late-onset Alzheimer's disease (LOAD). However, 50 percent of Alzheimer's disease cases do not carry an APOE4 allele, suggesting that other risk factors must exist. We performed a two-stage genome-wide screen in sibling pairs with LOAD to detect other susceptibility loci. Here we report evidence for an Alzheimer's disease locus on chromosome 10. Our stage one multipoint lod score (logarithm of the odds ratio for linkage/no linkage) of 2.48 (266 sibling pairs) increased to 3.83 in stage 2 (429 sibling pairs) close to D10S1225 (79 centimorgans). This locus modifies risk for Alzheimer's disease independent of APOE genotype.

Ethical aspects of research into Alzheimer disease. A European Delphi Study focused on genetic and non-genetic research.

BACKGROUND: Although genetic research into Alzheimer disease (AD) is increasing, the ethical aspects of this kind of research and the differences between ethical issues related to genetic and non-genetic research into AD have not yet received much attention. OBJECTIVES: (1) To identify and compare the five ethical issues considered most important by surveyed expert panellists in non-genetic and genetic AD research and (2) to compare our empirical findings with ethical issues in genetic research in general as described in the literature. METHOD: A modified Delphi study in two rounds RESULTS: Genetic and non-genetic research into AD generated an approximately equal number of topics with a considerable overlap. Different priorities in the ethics of both types of research were found. Genetic research raised new topics such as "confidentiality of genetic information" and "implications of research for relatives" which changes the impact and application of existing ethical topics such as "informed consent" and is judged to have more impact on both individuals and society. A difference with the results of more theoretical approaches on ethical aspects related to AD research was also found. CONCLUSIONS: Different priorities are given to ethical issues in genetic and non-genetic research. These arise partly because genetic research causes unique and new questions, mostly related to the position of family members and the status of and access to genetic information. Differences found between the results of our empirical study and the more theoretical literature, suggest an additional value for empirical research in medical ethics.

CSF evidence of pericyte damage in Alzheimer's disease is associated with markers of blood-brain barrier dysfunction and disease pathology.

BACKGROUND: We aimed to assess the relationship between levels of a cerebrospinal fluid (CSF) marker of pericyte damage, soluble platelet-derived growth factor receptor ß (sPDGFRß) and CSF markers of blood-brain barrier (BBB) integrity (CSF albumin and CSF/serum albumin ratio) and disease pathology (reduced CSF Aß42 and elevated CSF total and phosphorylated tau) in Alzheimer's disease (AD). METHODS: sPDGFRß and albumin were measured by sandwich ELISA in ante-mortem CSF from 39 AD and 39 age-matched controls that were grouped according to their biomarker profile (i.e. AD cases t-tau > 400 pg/mL, p-tau > 60 pg/mL and Aß42 < 550 pg/mL). sPDGFRß was also measured in matched serum and CSF samples (n = 23) in a separate neurologically normal group for which the CSF/serum albumin ratio had been determined. RESULTS: CSF sPDGFRß level was significantly increased in AD (p = 0.0038) and correlated positively with albumin (r = 0.45, p = 0.007), total tau (r = 0.50, p = 0.0017) and phosphorylated tau (r = 0.41, p = 0.013) in AD but not in controls. CSF sPDGFRß did not correlate with Aß42. Serum and CSF sPDGFRß were positively correlated (r = 0.547, p = 0.0085) in the independent neurologically normal CSF/serum matched samples. CONCLUSIONS: We provide further evidence of an association between pericyte injury and BBB breakdown in AD and novel evidence that a CSF marker of pericyte injury is related to the severity of AD pathology.

MMP-2, -3 and -9 levels and activity are not related to Abeta load in the frontal cortex in Alzheimer's disease.

Matrix metalloproteinases (MMPs) -2, -3 and -9 are up-regulated in several cell types on exposure to amyloid beta peptide (Abeta) and have Abeta-degrading activity in vitro. The aims of this study were to determine (i) the distribution of MMP-2, -3 and -9 in the cerebral cortex in Alzheimer's disease (AD) and control brains; (ii) whether the levels and activity of these proteases are increased in AD; and (iii) whether their activity is related to Abeta load. In addition, we examined whether promoter polymorphisms in the MMP-3 and -9 genes are associated with AD in the study cohort. Paraffin sections of frontal lobe from AD and control cases were immunostained for MMP-2, -3 and -9 and tissue homogenates used for MMP activity assays. DNA from these cases was genotyped for the MMP-3 5A/6A (-1171) and MMP-9 C-1562T promoter polymorphisms. Immunohistochemistry revealed MMP-3 in plaques and both MMP-3 and -9 around scattered neurones. The levels and activity of all three MMPs were similar in AD and control brains and bore no relationship to Abeta load. Analysis of MMP-3 -1171 5A/6A allele frequencies showed that the 6A allele (with reduced promoter activity) was associated with AD; the MMP-9 C-1562T polymorphism was not. The levels and activities of MMP-2, -3 and -9 are not increased in the frontal cortex in AD and are not related to Abeta load. Our findings suggest that altered expression of these proteases does not make a significant contribution to the accumulation of Abeta in AD.

Angiotensin-converting enzyme (ACE) levels and activity in Alzheimer's disease, and relationship of perivascular ACE-1 to cerebral amyloid angiopathy.

AIMS: Several observations point to the involvement of angiotensin-converting enzyme-1 (ACE-1) in Alzheimer's disease (AD): ACE-1 cleaves amyloid-beta peptide (Abeta) in vitro, the level and activity of ACE-1 are reportedly increased in AD, and variations in the ACE-1 gene are associated with AD. We analysed ACE-1 activity and expression in AD and control brains, particularly in relation to Abeta load and cerebral amyloid angiopathy (CAA). METHODS: ACE-1 activity was measured in the frontal cortex from 58 control and 114 AD cases of known Abeta load and CAA severity. The distribution of ACE-1 was examined immunohistochemically. In five AD cases with absent or mild CAA, five with moderate to severe CAA and five controls with absent or mild CAA, levels of vascular ACE-1 were assessed by quantitative immunofluorescence. RESULTS: ACE-1 activity was increased in AD (P < 0.001) and correlated directly with parenchymal Abeta load (P = 0.05). Immunohistochemistry revealed ACE-1 in neurones and cortical blood vessels - in the intima but most abundant perivascularly. Cases with moderate to severe CAA had significantly more vessel-associated ACE-1 than did those with little or no CAA. Perivascular ACE-1 did not colocalize with Abeta, smooth muscle actin, glial fibrillary acidic protein, collagen IV, vimentin or laminin, but was similarly distributed to extracellular matrix (ECM) proteins fibronectin and decorin. CONCLUSIONS: Our findings indicate that ACE-1 activity is increased in AD, in direct relationship to parenchymal Abeta load. Increased ACE-1, probably of neuronal origin, accumulates perivascularly in severe CAA and colocalizes with vascular ECM. The possible relationship of ACE-1 to the deposition of perivascular ECM remains to be determined.

Behavioral effects of kappa-opioid-receptor stimulation on neonatal rats.

Previous results show that endogenous opioid systems mediate affective responses in neonatal rats. Opioids modulate isolation-induced ultrasonic vocalizations and analgesia. This study further examines the behavioral effects of kappa-receptor-system stimulation on 10-day-old rats. With the agonist U50,488, response to isolation in terms of vocalizations, activity levels, and pain sensitivity was tested. In contrast to morphine's effects (primarily a mu-agonist), the kappa-agonist U50,488 produced increased vocalizing and hyperactivity, although both opioid agonists caused analgesia. Isolation adds to the U50,488-mediated increase in the latency for paw withdrawal from heat. This study suggests that the kappa system provokes calling and activity as opposed to the quieting effects of mu-agonists found in previous studies. These differential effects may be due in part to the interaction of the opioid and dopamine systems.

Behaviorally functional opioid systems in infant rats: II. Evidence for pharmacological, physiological, and psychological mediation of pain and stress.

In order to determine the behavioral characteristics of the neonatal opioid system during distressful situations, a modification of the hot-plate paw-lick test used on adults was developed for infant rats. Ten-day-old pups were analgesic to heat following morphine administration. Pretreatment with an opioid antagonist prevented the analgesia. Morphine analgesia was significantly greater in pups group isolated from the dam. Saline control pups group isolated from the dam exhibited longer latencies than their nest-housed siblings. Individual isolation for 5 min increased paw-withdrawal latency markedly. This was also naltrexone reversible. This analgesia was not seen when pups were tested directly from the nest or when grouped with other pups for the 5 min. It is suggested that the opioid system(s) for stress and pain are functional in Day 10 rats and short-term isolation from the dam is a probable natural stressor that is modulated by endogenous opioid release.

Behaviorally functional opioid systems in infant rats: I. Evidence for olfactory and gustatory classical conditioning.

In order to characterize behavioral function of opioid systems in neonatal rats, two series of experiments were initiated. In one, the reinforcing properties of exogenous opioids were investigated in 5-day-old pups. Specifically, the infant's ability to associate the novel taste of saccharin, received while suckling, with ip morphine injections was assessed. Rats that received 0.5 ml of saccharin prior to morphine administration ingested considerably more saccharin on Day 10 than did control rats. The second set of experiments was conducted to determine whether rat pups could associate a novel odor with morphine administration. Five days after conditioning, that stimulus was highly preferred by morphine-treated pups compared with saline control pups. Thus positive associations were formed with either a novel taste stimulus experienced while suckling or with an odor experienced during social isolation. Conditioning was cue specific and was retained for at least 5 days. The formation of these associations was blocked with opioid antagonists given prior to conditioning. These data suggest behaviorally functional opioid receptors and raise the possibility of a functional role of the endogenous opioids in motivational processes in infant rats.

Ontogeny of noradrenergic effects on ultrasonic vocalizations in rat pups.

The ontogeny of noradrenergic effects and the interaction of opioid and noradrenergic systems on vocalizations in rat pups from Day 10 to Day 18 were evaluated. Day 10 pups given clonidine (0.05 or 0.5 mg/kg) ip showed a sustained high level of calling throughout a 25-min isolation period that was reversed with yohimbine (0.1 mg/kg). Day 15 pups showed identical profiles with a lower baseline rate. Day 17 pups' calls were differentially affected according to dose; Day 18 pups reduced vocalizing with clonidine. In addition, it was found that at all ages when clonidine increased calling during isolation, the pups vocalized in the nest as well. Naltrexone, an opioid antagonist, lost its effectiveness to increase vocalizations after Day 15 unless it was given subsequent to clonidine. These results suggest that pups' vocalizations are differentially affected by noradrenergic and opioid stimulation or inhibition with developmental changes.

Effect of isolation conditions on brain regional enkephalin and beta-endorphin levels and vocalizations in 10-day-old rat pups.

Young rat pups were isolated from their dams under different conditions. The endogenous opioid peptides were measured in brain regions after isolation. Because there is no uptake mechanism for peptides released at the synapse and because released peptide is rapidly degraded enzymatically, decreases in peptide levels over this time course can be interpreted as release from terminals. No change was observed in either peptide in the hypothalamus, septum, or amygdala after isolation compared with controls. Significant decreases were seen in the midbrain after isolation. A comparison of peptide levels and ultrasonic vocalizations in the pups isolated in familiar, novel, or control conditions was also performed. Enkephalin levels in the midbrain were decreased in familiar and novel conditions, but in the brainstem opioid peptides were decreased only in the familiar condition. The greater involvement of the opioid peptides in the pups isolated in familiar conditions may contribute to the ability of naltrexone to block vocalization.

Cocaine-induced effects on isolation stress in neonatal rats.

The effects of cocaine administration on isolation-induced vocalizations and activity levels in 10-day-old rat pups were examined. Day 10 pups given cocaine (1.25, 2.5, 5, 10, & 20 mg/kg ip) vocalized significantly less than their caffeine- (10 mg/kg) and saline-administered siblings during a 5-min isolation period. Cocaine- and caffeine-administered pups also demonstrated a significant increase in overall activity compared with controls. In addition, intraperitoneal administration of the dopamine antagonist haloperidol (0.5 & 1.0 mg/kg) before 1.25 and 2.5 mg/kg cocaine produced a significant elevation in vocalizations compared with saline pretreatment, which indicates a blocking of cocaine's effect on calling behavior. These results suggest that the endogenous dopamine system involved with reinforcement and reward may quell the stress of isolation in the infant rat.

Conditioned opioid release in ten-day-old rats.

Ten-day-old rats, for whom an orange scent predicted morphine injections at 5 days of age, exhibited a marked preference for orange that was fully naltrexone reversible. Moreover, such rats, when smelling orange during a heat-escape task, exhibited a higher pain threshold than control rats. Together, these findings suggest that the orange odor in conditioned rats caused a release of endogenous opioids that both sustained choice behavior and modulated pain systems.