Systemic infection exacerbates cerebrovascular dysfunction in Alzheimer's disease.

We studied the effects of systemic infection on brain cytokine level and cerebral vascular function in Alzheimer's disease and vascular dementia, in superior temporal cortex (Brodmann area 22) from Alzheimer's disease patients (n = 75), vascular dementia patients (n = 22) and age-matched control subjects (n = 46), stratified according to the presence or absence of terminal systemic infection. Brain cytokine levels were measured using Mesoscale Discovery Multiplex Assays and markers of cerebrovascular function were assessed by ELISA. Multiple brain cytokines were elevated in Alzheimer's disease and vascular dementia: IL-15 and IL-17A were maximally elevated in end-stage Alzheimer's disease (Braak tangle stage V-VI) whereas IL-2, IL-5, IL12p40 and IL-16 were highest in intermediate Braak tangle stage III-IV disease. Several cytokines (IL-1ß, IL-6, TNF-α, IL-8 and IL-15) were further raised in Alzheimer's disease with systemic infection. Cerebral hypoperfusion-indicated by decreased MAG:PLP1 and increased vascular endothelial growth factor-A (VEGF)-and blood-brain barrier leakiness, indicated by raised levels of fibrinogen, were exacerbated in Alzheimer's disease and vascular dementia patients, and also in non-dementia controls, with systemic infection. Amyloid-ß42 level did not vary with infection or in association with brain cytokine levels. In controls, cortical perfusion declined with increasing IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13 and tumour necrosis factor-α (TNF-α) but these relationships were lost with progression of Alzheimer's disease, and with infection (even in Braak stage 0-II brains). Cortical platelet-derived growth factor receptor-ß (PDGFRß), a pericyte marker, was reduced, and endothelin-1 (EDN1) level was increased in Alzheimer's disease; these were related to amyloid-ß level and disease progression and only modestly affected by systemic infection. Our findings indicate that systemic infection alters brain cytokine levels and exacerbates cerebral hypoperfusion and blood-brain barrier leakiness associated with Alzheimer's disease and vascular dementia, independently of the level of insoluble amyloid-ß, and highlight systemic infection as an important contributor to dementia, requiring early identification and treatment in the elderly population.

Small vessel disease, neurovascular regulation and cognitive impairment: post-mortem studies reveal a complex relationship, still poorly understood.

The contribution of vascular disease to cognitive impairment is under-recognized and the pathogenesis is poorly understood. This information gap has multiple causes, including a lack of post-mortem validation of clinical diagnoses of vascular cognitive impairment (VCI) or vascular dementia (VaD), the exclusion of cases with concomitant neurodegenerative disease when diagnosing VCI/VaD, and a lack of standardization of neuropathological assessment protocols for vascular disease. Other contributors include a focus on end-stage destructive lesions to the exclusion of more subtle types of diffuse brain injury, on structural abnormalities of arteries and arterioles to the exclusion of non-structural abnormalities and capillary damage, and the use of post-mortem sampling strategies that are biased towards the identification of neurodegenerative pathologies. Recent studies have demonstrated the value of detailed neuropathology in characterizing vascular contributions to cognitive impairment (e.g. in diabetes), and highlight the importance of diffuse white matter changes, capillary damage and vasoregulatory abnormalities in VCI/VaD. The use of standardized, evidence-based post-mortem assessment protocols and the inclusion of biochemical as well as morphological methods in neuropathological studies should improve the accuracy of determination of the contribution of vascular disease to cognitive impairment and clarify the relative contribution of different pathogenic processes to the tissue damage.

Cerebrovascular disease in ageing and Alzheimer's disease.

Cerebrovascular disease (CVD) and Alzheimer's disease (AD) have more in common than their association with ageing. They share risk factors and overlap neuropathologically. Most patients with AD have Aß amyloid angiopathy and degenerative changes affecting capillaries, and many have ischaemic parenchymal abnormalities. Structural vascular disease contributes to the ischaemic abnormalities in some patients with AD. However, the stereotyped progression of hypoperfusion in this disease, affecting first the precuneus and cingulate gyrus, then the frontal and temporal cortex and lastly the occipital cortex, suggests that other factors are more important, particularly in early disease. Whilst demand for oxygen and glucose falls in late disease, functional MRI, near infrared spectroscopy to measure the saturation of haemoglobin by oxygen, and biochemical analysis of myelin proteins with differential susceptibility to reduced oxygenation have all shown that the reduction in blood flow in AD is primarily a problem of inadequate blood supply, not reduced metabolic demand. Increasing evidence points to non-structural vascular dysfunction rather than structural abnormalities of vessel walls as the main cause of cerebral hypoperfusion in AD. Several mediators are probably responsible. One that is emerging as a major contributor is the vasoconstrictor endothelin-1 (EDN1). Whilst there is clearly an additive component to the clinical and pathological effects of hypoperfusion and AD, experimental and clinical observations suggest that the disease processes also interact mechanistically at a cellular level in a manner that exacerbates both. The elucidation of some of the mechanisms responsible for hypoperfusion in AD and for the interactions between CVD and AD has led to the identification of several novel therapeutic approaches that have the potential to ameliorate ischaemic damage and slow the progression of neurodegenerative disease.

Altered Gene Expression Within the Renin-Angiotensin System in Normal Aging and Dementia.

The renin-angiotensin system (RAS) is dysregulated in Alzheimer's disease (AD). In this study, we have explored the hypothesis that an -age--related imbalance in brain RAS is a trigger for RAS dysregulation in AD. We characterized RAS gene expression in the frontal cortex from (i) a cohort of normal aging (n = 99, age range = 19-96 years) and (ii) a case-control cohort (n = 209) including AD (n = 66), mixed dementia (VaD + AD; n = 50), pure vascular dementia (VaD; n = 42), and age-matched controls (n = 51). The AD, mixed dementia, and age-matched controls were further stratified by Braak tangle stage (BS): BS0-II (n = 48), BSIII-IV (n = 44), and BSV-VI (n = 85). Gene expression was calculated by quantitative PCR (qPCR) for ACE1, AGTR1, AGTR2, ACE2, LNPEP, and MAS1 using the 2-∆∆Cq method, after adjustment for reference genes (RPL13 and UBE2D2) and cell-specific calibrator genes (NEUN, GFAP, PECAM). ACE1 and AGTR1, markers of classical RAS signaling, and AGTR2 gene expression were elevated in normal aging and gene expression in markers of protective downstream regulatory RAS signaling, including ACE2, MAS1, and LNPEP, were unchanged. In AD and mixed dementia, AGTR1 and AGTR2 gene expression were elevated in BSIII-IV and BSV-VI, respectively. MAS1 gene expression was reduced at BSV-VI and was inversely related to parenchymal Aß and tau load. LNPEP gene expression was specifically elevated in VaD. These data provide novel insights into RAS signaling in normal aging and dementia.

A single nuclear transcriptomic characterisation of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer's disease.

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer's disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased ß-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD.

Elevated late-life blood pressure may maintain brain oxygenation and slow amyloid-ß accumulation at the expense of cerebral vascular damage.

Hypertension in midlife contributes to cognitive decline and is a modifiable risk factor for dementia. The relationship between late-life hypertension and dementia is less clear. We have investigated the relationship of blood pressure and hypertensive status during late life (after 65 years) to post-mortem markers of Alzheimer's disease (amyloid-ß and tau loads); arteriolosclerosis and cerebral amyloid angiopathy; and to biochemical measures of ante-mortem cerebral oxygenation (the myelin-associated glycoprotein:proteolipid protein-1 ratio, which is reduced in chronically hypoperfused brain tissue, and the level of vascular endothelial growth factor-A, which is upregulated by tissue hypoxia); blood-brain barrier damage (indicated by an increase in parenchymal fibrinogen); and pericyte content (platelet-derived growth factor receptor ß, which declines with pericyte loss), in Alzheimer's disease (n = 75), vascular (n = 20) and mixed dementia (n = 31) cohorts. Systolic and diastolic blood pressure measurements were obtained retrospectively from clinical records. Non-amyloid small vessel disease and cerebral amyloid angiopathy were scored semiquantitatively. Amyloid-ß and tau loads were assessed by field fraction measurement in immunolabelled sections of frontal and parietal lobes. Homogenates of frozen tissue from the contralateral frontal and parietal lobes (cortex and white matter) were used to measure markers of vascular function by enzyme-linked immunosorbent assay. Diastolic (but not systolic) blood pressure was associated with the preservation of cerebral oxygenation, correlating positively with the ratio of myelin-associated glycoprotein to proteolipid protein-1 and negatively with vascular endothelial growth factor-A in both the frontal and parietal cortices. Diastolic blood pressure correlated negatively with parenchymal amyloid-ß in the parietal cortex. In dementia cases, elevated late-life diastolic blood pressure was associated with more severe arteriolosclerosis and cerebral amyloid angiopathy, and diastolic blood pressure correlated positively with parenchymal fibrinogen, indicating blood-brain barrier breakdown in both regions of the cortex. Systolic blood pressure was related to lower platelet-derived growth factor receptor ß in controls in the frontal cortex and in dementia cases in the superficial white matter. We found no association between blood pressure and tau. Our findings demonstrate a complex relationship between late-life blood pressure, disease pathology and vascular function in dementia. We suggest that hypertension helps to reduce cerebral ischaemia (and may slow amyloid-ß accumulation) in the face of increasing cerebral vascular resistance, but exacerbates vascular pathology.

Age-related reduction in brain ACE-2 is not exacerbated by Alzheimer's disease pathology in mouse models of Alzheimer's disease.

An imbalance in the circulatory and organ-specific renin-angiotensin system (RAS) pathways is associated with age-related dysfunction and disease including cardiovascular burden and more recently Alzheimer's disease (AD). It is currently unclear whether an age-associated imbalance in components of the RAS within the brain precedes the onset of AD or whether a RAS imbalance is associated with the onset of disease pathology and cognitive decline. Angiotensin-converting enzyme-1 (ACE-1) and -2 (ACE-2) protein (ELISA) and enzyme activity (FRET assay), markers of the classical and counter-regulatory RAS axis respectively, and Ang-II and Ang-(1-7) peptide levels (ELISA), were measured in the left cortex across four transgenic AD mouse models of amyloid pathology (5xFAD - 2, 6, and 12 months of age; Apd9 - 3-4, 12, and 18 months of age; Tg2576 - 3-4 and 24 months of age; and PDAPP - 3-4, 7, 11, 15, and 18 months of age) and littermate wild-type (WT) controls. ACE-1 level, and enzyme activity, was unaltered in relation to age in WT mice and across all four models. In contrast, ACE-2 level and enzyme activity, was reduced and Ang-II increased with ageing in both WT animals and disease models. The changes in ACE-2 and Ang-II in AD models mirrored WT mice, except for the 5xFAD model, when the reduction in ACE-2 (and elevated Ang-II) was observed at a younger age. These data indicate an age-related dysregulation of brain RAS is likely to be driven by a reduction in ACE-2. The reduction in ACE-2 occurs at a young age, coinciding with early pathological changes and the initial deposition of Aß, and preceding neuronal loss and cognitive decline, in the transgenic AD models. However, the age-related loss was mirrored in WT mice suggesting that the change was independent of pathological Aß deposition.

Pathological changes within the cerebral vasculature in Alzheimer's disease: New perspectives.

Cerebrovascular disease underpins vascular dementia (VaD), but structural and functional changes to the cerebral vasculature contribute to disease pathology and cognitive decline in Alzheimer's disease (AD). In this review, we discuss the contribution of cerebral amyloid angiopathy and non-amyloid small vessel disease in AD, and the accompanying changes to the density, maintenance and remodelling of vessels (including alterations to the composition and function of the cerebrovascular basement membrane). We consider how abnormalities of the constituent cells of the neurovascular unit - particularly of endothelial cells and pericytes - and impairment of the blood-brain barrier (BBB) impact on the pathogenesis of AD. We also discuss how changes to the cerebral vasculature are likely to impair Aß clearance - both intra-periarteriolar drainage (IPAD) and transport of Aß peptides across the BBB, and how impaired neurovascular coupling and reduced blood flow in relation to metabolic demand increase amyloidogenic processing of APP and the production of Aß. We review the vasoactive properties of Aß peptides themselves, and the probable bi-directional relationship between vascular dysfunction and Aß accumulation in AD. Lastly, we discuss recent methodological advances in transcriptomics and imaging that have provided novel insights into vascular changes in AD, and recent advances in assessment of the retina that allow in vivo detection of vascular changes in the early stages of AD.

Dysregulation of ACE-1 in Normal Aging and the Early Stages of Alzheimer's Disease.

An imbalance in the renin-angiotensin system (RAS) is associated with cognitive decline and disease pathology in Alzheimer's disease (AD). In this study, we have investigated changes in the brain angiotensin-converting enzyme-1 (ACE-1) and angiotensin-II (Ang-II), and the counter-regulatory angiotensin-converting enzyme-2 (ACE-2), in the frontal and temporal cortex during normal aging and in the early stages of AD. We studied a cohort of normal aging (n = 121; 19-95 years age-at-death) from the Sudden Death Brain Bank, University of Edinburgh, United Kingdom, and AD and age-matched controls (n = 60) from the South West Dementia Brain Bank, University of Bristol, United Kingdom, stratified according to Braak tangle stage (BS): 0-II, III-IV (intermediate disease), and V-VI (end-stage disease). ACE-1 and ACE-2 enzyme activity were measured using fluorogenic peptide activity assays. ACE-1, ACE-2, and Ang-II protein level were measured by enzyme-linked immunosorbent assay (ELISA). In both regions, ACE-1 protein and Ang-II levels correlated positively with age whereas ACE-1 enzyme activity was inversely related to age. ACE-1 protein correlated positively with Ang-II, whilst ACE-1 activity correlated inversely with Ang-II in normal aging. ACE-1 enzyme activity was elevated at an early/intermediate stage, BS III-IV compared to BS 0-II in the temporal cortex in AD. ACE-2 protein and enzyme activity were unchanged with aging and in AD. In conclusion, ACE-1 activity is induced in the early stages of AD independently from normal physiological age-related changes in ACE-1 protein.

Pericyte Contractile Responses to Endothelin-1 and Aß Peptides: Assessment by Electrical Impedance Assay.

Pericytes are vascular mural cells that contract and relax in response to vasoactive stimuli to regulate neurovascular coupling and cerebral blood flow. Pericytes are damaged and degenerate in Alzheimer's disease (AD). We previously showed that the level of the regulatory vasoconstrictor, endothelin-1 (EDN1), is elevated in AD cerebral cortex and upregulated by amyloid-beta (Aß). We have used electrical impedance analysis to monitor the contractile and proliferative response of cultured human fetal and adult brain-derived pericytes to EDN1 in real-time. EDN1 caused transient, dose-dependent contraction of fetal and adult brain pericytes that was mediated by EDN1 type A receptors and increased the subsequent proliferation of fetal but not adult cells. The contractile responses to EDN1 were weaker in the adult pericytes. The EDN1-mediated contractile response of fetal pericytes was unchanged after exposure to Aß1 - 40 or Aß1 - 42 (0.1-10 µM) for 1 h but both contraction and subsequent relaxation were significantly impaired upon exposure to Aß for 24 h. These data suggest that chronic exposure to Aß interferes with EDN1-mediated pericyte contractility, potentially contributing to neurovascular uncoupling and reduced cerebral blood flow in AD.

Mediators of cerebral hypoperfusion and blood-brain barrier leakiness in Alzheimer's disease, vascular dementia and mixed dementia.

In vascular dementia (VaD) and Alzheimer's disease (AD), cerebral hypoperfusion and blood-brain barrier (BBB) leakiness contribute to brain damage. In this study, we have measured biochemical markers and mediators of cerebral hypoperfusion and BBB in the frontal (BA6) and parietal (BA7) cortex and underlying white matter, to investigate the pathophysiology of vascular dysfunction in AD, VaD and mixed dementia. The ratio of myelin-associated glycoprotein to proteolipid protein-1 (MAG:PLP1), a post-mortem biochemical indicator of the adequacy of ante-mortem cerebral perfusion; the concentration of fibrinogen adjusted for haemoglobin level, a marker of blood-brain barrier (BBB) leakiness; the level of vascular endothelial growth factor-A (VEGF), a marker of tissue hypoxia; and endothelin-1 (EDN1), a potent vasoconstrictor, were measured by ELISA in the frontal and parietal cortex and underlying white matter in 94 AD, 20 VaD, 33 mixed dementia cases and 58 age-matched controls. All cases were assessed neuropathologically for small vessel disease (SVD), cerebral amyloid angiopathy (CAA) severity, Aß and phospho-tau parenchymal load, and Braak tangle stage. Aß40 and Aß42 were measured by ELISA in guanidine-HCl tissue extracts. We found biochemical evidence of cerebral hypoperfusion in AD, VaD and mixed dementia to be associated with SVD, Aß level, plaque load, EDN1 level and Braak tangle stage, and to be most widespread in mixed dementia. There was evidence of BBB leakiness in AD-limited to the cerebral cortex and related to EDN1 level. In conclusion, abnormalities of cerebral perfusion and BBB function in common types of dementia can largely be explained by a combination of arteriolosclerosis, and Aß-, tau- and endothelin-related vascular dysfunction. The relative contributions of these processes vary considerably both between and within the diseases.

Differing associations between Aß accumulation, hypoperfusion, blood-brain barrier dysfunction and loss of PDGFRB pericyte marker in the precuneus and parietal white matter in Alzheimer's disease.

Recent studies implicate loss of pericytes in hypoperfusion and blood-brain barrier (BBB) leakage in Alzheimer's disease (AD). In this study, we have measured levels of the pericyte marker, platelet-derived growth factor receptor-ß (PDGFRB), and fibrinogen (to assess blood-brain barrier leakage), and analyzed their relationship to indicators of microvessel density (von Willebrand factor level), ante-mortem oxygenation (myelin-associated glycoprotein:proteolipid protein-1 ratio and vascular endothelial growth factor level), Aß level and plaque load, in precuneus and underlying white matter from 49 AD to 37 control brains. There was reduction in PDGFRB and increased fibrinogen in the precuneus in AD. These changes correlated with reduction in oxygenation and with plaque load. In the underlying white matter, increased fibrinogen correlated with reduced oxygenation, but PDGFRB level was unchanged. The level of platelet-derived growth factor-ßß (PDGF-BB), important for pericyte maintenance, was increased in AD but mainly in the insoluble tissue fraction, correlating with insoluble Aß level. Loss of the PDGFRB within the precuneus in AD is associated with fibrinogen leakage and reduced oxygenation, and related to fibrillar Aß accumulation. In contrast, fibrinogen leakage and reduced oxygenation of underlying white matter occur independently of loss of PDGFRB, perhaps secondary to reduced transcortical perfusion.

Clusterin levels are increased in Alzheimer's disease and influence the regional distribution of Aß.

Clusterin, also known as apoJ, is a lipoprotein abundantly expressed within the CNS. It regulates Aß fibril formation and toxicity and facilitates amyloid-ß (Aß) transport across the blood-brain barrier. Genome-wide association studies have shown variations in the clusterin gene (CLU) to influence the risk of developing sporadic Alzheimer's disease (AD). To explore whether clusterin modulates the regional deposition of Aß, we measured levels of soluble (NP40-extracted) and insoluble (guanidine-HCl-extracted) clusterin, Aß40 and Aß42 by sandwich ELISA in brain regions with a predilection for amyloid pathology-mid-frontal cortex (MF), cingulate cortex (CC), parahippocampal cortex (PH), and regions with little or no pathology-thalamus (TH) and white matter (WM). Clusterin level was highest in regions with plaque pathology (MF, CC, PH and PC), approximately mirroring the regional distribution of Aß. It was significantly higher in AD than controls, and correlated positively with Aß42 and insoluble Aß40. Soluble clusterin level rose significantly with severity of cerebral amyloid angiopathy, and in MF and PC regions was highest in APOE ɛ4 homozygotes. In the TH and WM (areas with little amyloid pathology) clusterin was unaltered in AD and did not correlate with Aß level. There was a significant positive correlation between the concentration of clusterin and the regional levels of insoluble Aß42; however, the molar ratio of clusterin : Aß42 declined with insoluble Aß42 level in a region-dependent manner, being lowest in regions with predilection for Aß plaque pathology. Under physiological conditions, clusterin reduces aggregation and promotes clearance of Aß. Our findings indicate that in AD, clusterin increases, particularly in regions with most abundant Aß, but because the increase does not match the rising level of Aß42, the molar ratio of clusterin : Aß42 in those regions falls, probably contributing to Aß deposition within the tissue.

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

Hyperactivity of the renin-angiotensin system (RAS) is associated with the pathogenesis of Alzheimer's 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, Aß 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 Aß 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.

Cerebral Hypoperfusion and the Energy Deficit in Alzheimer's Disease.

There is a perfusion deficit in Alzheimer's disease (AD), commencing in the precuneus and spreading to other parts of the cerebral cortex. The deficit anticipates the development of dementia, contributes to brain damage, and is caused by both functional and structural abnormalities of the cerebral vasculature. Most of the abnormalities are probably secondary to the accumulation of Aß but the consequent hypoperfusion may, in turn, increase Aß production. In the early stages of disease, abnormalities that cause vasoconstriction predominate. These include cholinergic vascular denervation, inhibition of endothelial nitric oxide synthase, increased production of endothelin-1 production and possibly also of angiotensin II. Patients with AD also have an increased prevalence of structural disease of cerebral microvessels, particularly CAA and capillary damage, and particularly in the later stages of disease these are likely to make an important contribution to the cerebral hypoperfusion. The metabolic abnormalities that cause early vascular dysfunction offer several targets for therapeutic intervention. However, for intervention to be effective it probably needs to be early. Prolonged cerebral hypoperfusion may induce compensatory circulatory changes that are themselves damaging, including hypertension and small vessel disease. This has implications for the use of antihypertensive drugs once there is accumulation of Aß within the brain.

Pathophysiology of Hypoperfusion of the Precuneus in Early Alzheimer's Disease.

The earliest decline in cerebral perfusion in Alzheimer's disease (AD) is in the medial parietal cortex (precuneus). We have analyzed precuneus in post-mortem tissue from 70 AD and 37 control brains to explore the pathophysiology of the hypoperfusion: the contribution of arteriolosclerotic small vessel disease (SVD) and cerebral amyloid angiopathy (CAA), and of the vasoconstrictors endothelin-1 (EDN1) and angiotensin II (Ang II), and the association with Aß. The myelin-associated glycoprotein:proteolipid protein-1 ratio (MAG:PLP1) was used as an indicator of oxygenation of the precuneus prior to death. MAG:PLP1 was reduced ∼50% in early AD (Braak stage III-IV). Although MAG:PLP1 remained low in advanced AD (stage V-VI), the reduction was less pronounced, possibly reflecting falling oxygen demand. Reduction in cortical MAG:PLP1 correlated with elevation in vascular endothelial growth factor (VEGF), another marker of hypoperfusion. Cortical MAG:PLP1 declined nonsignificantly with increasing SVD and CAA, but significantly with the concentration of EDN1, which was elevated approximately 75% in AD. In contrast, with reduction in cortical MAG:PLP1, Ang II level and angiotensin-converting enzyme (ACE) activity declined, showing a normal physiological response to hypoperfusion. MAG:PLP1 was reduced in the parietal white matter (WM) in AD but here the decline correlated positively (ie, physiologically) with WM EDN1. However, the decline of MAG:PLP1 in the WM was associated with increasing cortical EDN1 and perhaps reflected vasoconstriction of perforating arterioles, which traverse the cortex to perfuse the WM. EDN1 in the cortex correlated highly significantly with both soluble and insoluble Aß42, shown previously to upregulate neuronal endothelin-converting enzyme-2 (ECE2), but not with Aß40. Our findings demonstrate reduced oxygenation of the precuneus in early AD and suggest that elevated EDN1, resulting from Aß42-mediated upregulation of ECE2, is a contributor.

Angiotensin-converting enzyme 2 is reduced in Alzheimer's disease in association with increasing amyloid-ß and tau pathology.

BACKGROUND: Hyperactivity of the classical axis of the renin-angiotensin system (RAS), mediated by angiotensin II (Ang II) activation of the angiotensin II type 1 receptor (AT1R), is implicated in the pathogenesis of Alzheimer's disease (AD). Angiotensin-converting enzyme-2 (ACE-2) degrades Ang II to angiotensin 1-7 (Ang (1-7)) and counter-regulates the classical axis of RAS. We have investigated the expression and distribution of ACE-2 in post-mortem human brain tissue in relation to AD pathology and classical RAS axis activity. METHODS: We measured ACE-2 activity by fluorogenic peptide substrate assay in mid-frontal cortex (Brodmann area 9) in a cohort of AD (n = 90) and age-matched non-demented controls (n = 59) for which we have previous data on ACE-1 activity, amyloid ß (Aß) level and tau pathology, as well as known ACE1 (rs1799752) indel polymorphism, apolipoprotein E (APOE) genotype, and cerebral amyloid angiopathy severity scores. RESULTS: ACE-2 activity was significantly reduced in AD compared with age-matched controls (P < 0.0001) and correlated inversely with levels of Aß (r = -0.267, P < 0.001) and phosphorylated tau (p-tau) pathology (r = -0.327, P < 0.01). ACE-2 was reduced in individuals possessing an APOE ε4 allele (P < 0.05) and was associated with ACE1 indel polymorphism (P < 0.05), with lower ACE-2 activity in individuals homozygous for the ACE1 insertion AD risk allele. ACE-2 activity correlated inversely with ACE-1 activity (r = -0.453, P < 0.0001), and the ratio of ACE-1 to ACE-2 was significantly elevated in AD (P < 0.0001). Finally, we show that the ratio of Ang II to Ang (1-7) (a proxy measure of ACE-2 activity indicating  conversion of Ang II to Ang (1-7)) is reduced in AD. CONCLUSIONS: Together, our findings indicate that ACE-2 activity is reduced in AD and is an important regulator of the central classical ACE-1/Ang II/AT1R axis of RAS, and also that dysregulation of this pathway likely plays a significant role in the pathogenesis of AD.

White matter hypoperfusion and damage in dementia: post-mortem assessment.

Neuroimaging has revealed a range of white matter abnormalities that are common in dementia, some that predict cognitive decline. The abnormalities may result from structural diseases of the cerebral vasculature, such as arteriolosclerosis and amyloid angiopathy, but can also be caused by nonstructural vascular abnormalities (eg, of vascular contractility or permeability), neurovascular instability or extracranial cardiac or vascular disease. Conventional histopathological assessment of the white matter has tended to conflate morphological vascular abnormalities with changes that reflect altered interstitial fluid dynamics or white matter ischemic damage, even though the latter may be of extracranial or nonstructural etiology. However, histopathology is being supplemented by biochemical approaches, including the measurement of proteins involved in the molecular responses to brain ischemia, myelin proteins differentially susceptible to ischemic damage, vessel-associated proteins that allow rapid measurement of microvessel density, markers of blood-brain barrier dysfunction and axonal injury, and mediators of white matter damage. By combining neuroimaging with histopathology and biochemical analysis, we can provide reproducible, quantitative data on the severity of white matter damage, and information on its etiology and pathogenesis. Together these have the potential to inform and improve treatment, particularly in forms of dementia to which white matter hypoperfusion makes a significant contribution.

Accumulation of α-synuclein in dementia with Lewy bodies is associated with decline in the α-synuclein-degrading enzymes kallikrein-6 and calpain-1.

Kallikrein-6 and calpain-1 are amongst a small group of proteases that degrade α-synuclein. We have explored the possibility that reduction in the level or activity of these enzymes contributes to the accumulation of α-synuclein in Lewy body diseases. We measured calpain-1 activity by fluorogenic activity assay, kallikrein-6 level by sandwich ELISA, and levels of α-synuclein and α-synuclein phosphorylated at serine 129 (α-synuclein-P129), in post-mortem brain tissue in pure dementia with Lewy bodies (DLB, n=12), Alzheimer's disease (AD, n=20) and age-matched controls (n=19). Calpain-1 activity was significantly reduced in DLB within the cingulate and parahippocampal cortex, regions with highest α-synuclein and α-synuclein-P129 load, and correlated inversely with the levels of α-synuclein and α-synuclein-P129. Calpain-1 was unaltered in the thalamus and frontal cortex, regions with less α-synuclein pathology. Kallikrein-6 level was reduced in the cingulate cortex in the DLB cohort, and correlated inversely with α-synuclein and α-synuclein-P129. Kallikrein-6 was also reduced in DLB in the thalamus but not in relation to α-synuclein or α-synuclein-P129 load and was unaltered in the frontal and parahippocampal cortex. In SH-SY5Y cells overexpressing wild-type α-synuclein there was partial co-localisation of kallikrein-6 and calpain-1 with α-synuclein, and siRNA-mediated knock-down of kallikrein-6 and calpain-1 increased the amount of α-synuclein in cell lysates. Our results indicate that reductions in kallikrein-6 and calpain-1 may contribute to the accumulation of α-synuclein in DLB.