Laura E. Palmer

Abeta-degrading enzymes in Alzheimer's disease.

In Alzheimers disease (AD) Aβ accumulates because of imbalance between the production of Aβ and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Aβ is partly, if not in some cases solely, because of defects in its removal—mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Aβ. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Aβ‐degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin‐degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Aβin vitro or in animal studies include plasmin; endothelin‐converting enzymes ECE‐1 and ‐2; matrix metalloproteinases MMP‐2, ‐3 and ‐9; and angiotensin‐converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE‐2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEε4. We found no change in the level or activity of MMP‐2, ‐3 or ‐9 in AD. The level and activity of ACE are increased, the level being directly related to Aβ plaque load. Up‐regulation of some Aβ‐degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Aβ‐clearance pathways, reductions in the activity of particular Aβ‐degrading enzymes may become critical, leading to the development of AD and CAA.

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 Alzheimers disease (AD): ACE‐1 cleaves amyloid‐β peptide (Aβ) 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 Aβ 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 Aβ 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 Aβ 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 Aβ, 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 Aβ 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.

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

Clusterin mRNA and protein in Alzheimer's disease

Clusterin, a multifunctional lipoprotein is expressed in a number of tissues but expression is particularly high in the brain, where it binds to amyloid-β (Aβ), possibly facilitating Aβ transport into the bloodstream. Its concentration in peripheral blood was identified as a potential biomarker for Alzheimers disease (AD) and predicted retention of (11)C-Pittsburgh Compound B in the temporal lobe. Single-nucleotide polymorphisms in the clusterin gene, CLU, are associated with the risk of developing AD. We measured clusterin mRNA levels in control and AD brains and investigated the relationship of the clusterin protein to soluble, insoluble, and plaque-associated Aβ. Clusterin mRNA levels were unchanged when normalized to GAPDH but modestly increased in the frontal and temporal cortex in AD in relation to NSE and MAP-2. Levels of NSE and MAP-2 mRNA were reduced in the AD frontal cortex. Clusterin protein concentration was unchanged and did not correlate with the amount of Aβ present. In the frontal cortex, clusterin concentration was higher in APOE ε4-negative brains but no effect of APOE was detected in the temporal cortex or thalamus. Overall clusterin mRNA and protein levels are unaltered in the neocortex in AD and clusterin concentration does not reflect Aβ content. The increase in clusterin noted in peripheral blood in AD may reflect increased passage of this chaperone protein across the blood-brain barrier but further work is needed to determine how CLU variants influence the development of AD.

Neither sequence variation in the IL-10 gene promoter nor presence of IL-10 protein in the cerebral cortex is associated with Alzheimer's disease.

Interleukin 10 (IL-10) is an important anti-inflammatory cytokine produced in response to neuroinflammation and might be involved in modulating the progression of Alzheimers disease (AD) through inhibiting the action of pro-inflammatory cytokines. We have used immunohistochemistry, Western blotting, real time-PCR (RT-PCR) on frontal (BA 6/24) and temporal (BA 20-22) neocortex and hippocampus from AD and control brains as well as genetic association analysis to address the possible involvement of IL-10 in AD. Expression of IL-10 in AD and control brains at both protein and mRNA levels were detected. However, the level of expression, particularly of IL-10 protein, varied considerably in individual brains and we did not find a significant difference between AD and controls. Using direct sequencing we examined five single nucleotide polymorphisms (SNPs) (-3538, -1354, -1087, -824, -597) and two microsatellites (IL-10-G, IL-10-R) in the promoter region of the IL-10 gene. None of the identified SNPs were found to be associated with AD either individually or as haplotypes. Levels of IL-10 protein and gene expression examined also did not appear to be related to AD. Despite this being a relatively small sample, these data suggest that IL-10 does not play a major role in the development of AD.

Angiotensin-III is Increased in Alzheimer's Disease in Association with Amyloid-β and Tau Pathology

Hyperactivity of the renin-angiotensin system (RAS) is associated with the pathogenesis of Alzheimers disease (AD) believed to be mediated by angiotensin-II (Ang-II) activation of the angiotensin type 1 receptor (AT1R). We previously showed that angiotensin-converting enzyme-1 (ACE-1) activity, the rate-limiting enzyme in the production of Ang-II, is increased in human postmortem brain tissue in AD. Angiotensin-III (Ang-III) activates the AT1R and angiotensin type-2 receptor (AT2R), but its potential role in the pathophysiology of AD remains unexplored. We measured Ang-II and Ang-III levels by ELISA, and the levels and activities of aminopeptidase-A (AP-A) and aminopeptidase-N (AP-N) (responsible for the production and metabolism of Ang-III, respectively) in human postmortem brain tissue in the mid-frontal cortex (Brodmann area 9) in a cohort of AD (n = 90) and age-matched non-demented controls (n = 59), for which we had previous measurements of ACE-1 activity, Aβ level, and tau pathology (also in the mid-frontal cortex). We found that both Ang-II and Ang-III levels were significantly higher in AD compared to age-matched controls and that Ang-III, rather than Ang-II, was strongly associated with Aβ load and tau load. Levels of AP-A were significantly reduced in AD but AP-A enzyme activity was unchanged whereas AP-N activity was reduced in AD but AP-N protein level was unchanged. Together, these data indicate that the APA/Ang-III/APN/Ang-IV/AT4R pathway is dysregulated and that elevated Ang-III could contribute to the pathogenesis of AD.

SYMPOSIUM: Clearance of Aβ from the Brain in Alzheimer's Disease: Aβ‐Degrading Enzymes in Alzheimer's Disease

In Alzheimers disease (AD) Aβ accumulates because of imbalance between the production of Aβ and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Aβ is partly, if not in some cases solely, because of defects in its removal—mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Aβ. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Aβ‐degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin‐degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Aβin vitro or in animal studies include plasmin; endothelin‐converting enzymes ECE‐1 and ‐2; matrix metalloproteinases MMP‐2, ‐3 and ‐9; and angiotensin‐converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE‐2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEε4. We found no change in the level or activity of MMP‐2, ‐3 or ‐9 in AD. The level and activity of ACE are increased, the level being directly related to Aβ plaque load. Up‐regulation of some Aβ‐degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Aβ‐clearance pathways, reductions in the activity of particular Aβ‐degrading enzymes may become critical, leading to the development of AD and CAA.

Extended post-mortem delay times should not be viewed as a deterrent to the scientific investigation of human brain tissue: a study from the Brains for Dementia Research Network Neuropathology Study Group, UK

The study encompassed 556 brains, consecutively acquired through the Brains for Dementia Research Network between 2013 and 2016. Five Centres participated, Bristol (52 brains), Manchester (93 brains), Newcastle (76 brains), Oxford (213 brains) and Kings College, London (122 brains). These comprised 300 males (mean age 76.6 ± 12.8, range 6–104 years) and 256 females (mean age 80.3 ± 14.2, range 8–102 years). PMD varied from 6 to 279 h (mean 59.4 ± 32.9 h). Brains were divided along the mid-line. One hemi-brain was dissected fresh into coronal slices for pH measurements and frozen tissue sampling, while the other hemi-brain was placed whole into 10 % buffered formalin for diagnostic investigations. Brain pH was measured on the freshly sliced cerebral hemispheres at each of the five Centres at four standard locations—frontal lobe white matter anterior to the genu of the corpus callosum, occipito-parietal white matter posterior to the splenium, occipital pole white matter and cerebellar white matter—using the same pH meter in each Centre (Hanna Instruments Skin pH Portable Meter—HI99181). RIN was measured in frontal cortex (London, 89 cases) or cerebellum (Oxford, 118 cases). Briefly, RNA was extracted manually from frozen cerebellum using Allprep DNA/RNA minikit (Qiagen); RNA concentration was measured with Nanodrop 2000c; RIN was measured using Agilent 2100 Bioanalyzer. PMD was calculated as the interval between time of death and time when the dissected brain was placed in the freezer/into formalin. The relationships between, pH and PMD were analysed on all 556 cases—and when stratified according to agonal status in terms of sudden versus protracted death, and the presence or absence of sepsis at time of death, where this could be reliably determined from clinical notes or death certification. Sudden death involved conditions such as cardiac arrest and pulmonary embolism, whereas protracted death mostly involved carcinomatosis Although animal and cellular models can recapitulate certain aspects of the pathology and pathogenesis of human neurodegenerative diseases such as Alzheimer’s disease, frontotemporal lobar degeneration and Parkinson’s disease, none of these models is perfect. The study of human postmortem tissues is still essential for investigation of neurodegenerative disorders. It is a common perception that the long post-mortem delays (PMDs) that often limit the acquisition of brains after death are detrimental to brain quality, and therefore, prejudicial to the reliability of scientific data based on such materials. However, this viewpoint is largely anecdotal and has not been supported by scientific evidence. We have sought to address this issue by investigating how putative measures of tissue quality such as brain pH or values of RNA integrity (RIN) relate to PMD.

Erratum to “TNFR-associated factor-2 (TRAF-2) in Alzheimer’s disease” [Culpan et al. (Neurobiol Aging 2009 July;30(7):1052-60)]

Doris Culpan a,∗, Dougal Cram a, Kate Chalmers a, Abigail Cornish a, Laura Palmer a, Jennifer Palmer a, Anthony Hughes a, Peter Passmore b, David Craig b, Gordon K. Wilcock a, Patrick G. Kehoe a, Seth Love a a Dementia Research Group, Institute of Clinical Neurosciences, Department of Clinical Sciences at North Bristol, University of Bristol, John James Buildings, Frenchay Hospital, Frenchay, Bristol BS16 1LE, United Kingdom b Department of Geriatric Medicine, Queen’s University, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom

AN INTEGRATED APPROACH TO U.K. BRAIN BANKING ALLOWING RESEARCHERS TO SELECT TISSUE WITH PRECISION: MRC U.K. BRAIN BANK NETWORK, CSOLS TRACKING SYSTEM AND BRAINS FOR DEMENTIA RESEARCH

Results: The longitudinal and transitional nature of a readiness cohort creates the risk of the cohort and its concatenated trials becoming a ‘fish trap’ for its participants. With every step further into such a research project, it becomes more difficult for a participant to go back or retract from participation. Therefore, we recommend an adapted stagedconsent model for concatenated projects like EPAD, which are extended over time and multi-staged, and in which participants and data move from one stage to the next. This consent model feeds relevant information, bit by bit, along research participants’ journey, and asks informed consent at every moment in which important decisions need to be made by participants. Although informed consent is always given for a specific stage of the research project, information about the ‘totality of the project’ must always and explicitly be part of the informed consent process. Conclusions: It is critical to the success of readiness cohorts to carefully align ethical guidance for recruitment and informed consent. The ELSIwork package of EPAD recommends an adapted staged-consent model. This model may also be of value to other research collaborations in the process of developing and using readiness cohorts in Alzheimer’s disease research.