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.

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.

Exploring the putative role of kallikrein-6, calpain-1 and cathepsin-D in the proteolytic degradation of α-synuclein in multiple system atrophy.

AIMS: There is evidence that accumulation of α-synuclein (α-syn) in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) results from impaired removal of α-syn rather than its overproduction. Kallikrein-6 (KLK6), calpain-1 (CAPN1) and cathepsin-D (CTSD) are among a small number of proteases that cleave α-syn and are dysregulated in PD and DLB. Our aim in this study was to determine whether protease activity is altered in another α-synucleinopathy, multiple system atrophy (MSA), and might thereby modulate the regional distribution of α-syn accumulation. METHODS: mRNA and protein level and/or activity of KLK6, CAPN1 and CTSD were measured and assessed in relation to α-syn load in multiple brain regions (posterior frontal cortex, caudate nucleus, putamen, occipital cortex, pontine base and cerebellar white matter), in MSA (n = 20) and age-matched postmortem control tissue (n = 20). RESULTS: CTSD activity was elevated in MSA in the pontine base and cerebellar white matter. KLK6 and CAPN1 levels were elevated in MSA in the putamen and cerebellar white matter. However, the activity or level of these proteolytic enzymes did not correlate with the regional distribution of α-syn. CONCLUSIONS: Accumulation of α-syn in MSA is not due to reduced activity of the proteases we have studied. We suggest that their upregulation is likely to be a compensatory response to increased α-syn in MSA.

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.

Caveolin-1 and -2 and their relationship to cerebral amyloid angiopathy in Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) affects over 90% of patients with Alzheimer's disease (AD) and increases the risk of cerebral haemorrhage and infarction. Caveolae--cholesterol-enriched plasmalemmal microinvaginations--are implicated in the production of amyloid beta peptide (Abeta). Caveolin-1 (CAV-1) is essential for the formation of caveolae. Caveolin-2 (CAV-2) is expressed at the plasma membrane only when in a stable hetero-oligomeric complex with CAV-1. CAV-1 and CAV-2 are highly co-expressed by endothelium and smooth muscle. Recent studies suggest that down-regulation of CAV-1 causes a reduction in alpha-secretase activity and consequent accumulation of Abeta. We have used quantitative immunohistochemical techniques to assess the relationship between CAV-1 and CAV-2 with respect to Abeta accumulation in the cerebral vasculature in a series of post mortem brains. CAV-1 and CAV-2 were co-expressed within the tunica media and endothelium of cerebral blood vessels. There were regional differences in CAV-1 immunolabelling, which was significantly greater in the frontal cortex and white matter than in the parietal lobe (in both control and AD cases) or the temporal lobe (in AD alone). However, CAV-1 labelling in AD did not differ from that in controls in any of the three lobes examined. Assessment of CAV-1 labelling in relation to the severity of CAA showed CAV-1 to be significantly increased in the frontal white matter in a subgroup of AD cases with absent/mild CAA compared with controls with absent/mild CAA and to AD cases with moderate/severe CAA, but the latter groups did not show significant differences from one another. CAV-1 labelling did not vary with age, gender, APOE genotype, post mortem delay or brain weight. Only segments of blood vessels with particularly abundant Abeta and extensive loss of smooth muscle actin showed loss of CAV-1 and CAV-2 from the tunica media. Within these vessels endothelial CAV-1 was preserved and discontinuous CAV-2 labelling was noted along the outer aspect of the vessel wall. Our findings suggest that alterations in the expression of vascular CAV-1 and CAV-2 are unlikely to play a role in the development of CAA in AD.

Role of CD4+, CD8+ and double negative T-cells in the protection of SCID/beige mice against respiratory challenge with Rhodococcus equi.

To evaluate the contributions of T-lymphocyte subsets in pulmonary immunity against Rhodococcus equi, C.B-17 SCID/beige mice were adoptively transferred with splenic lymphocytes from congenic BALB/c mice previously infected with R. equi. Spleen cells were enriched for either CD4+ or CD8+ populations before inoculation, Flow cytometry showed that each enriched population contained less than 0.5% cross contamination. Groups of adoptively transferred SCID/beige mice were sacrificed 6 and 13 d after intranasal infection with R. equi. Bacterial clearance was measured in the lungs, liver and spleen. Lesion development was assessed by gross and histopathological score and the fate of transferred cells assessed by flow cytometry and by immunohistochemistry. SCID/beige mice receiving either CD4+ or CD8+ T-cells were able to clear the infection better than control mice. On d 6 post-infection, bacterial numbers were significantly lower in the lungs of CD4+ transferred mice as compared to CD8+ mice. By d 13, both groups had cleared R. equi from all organs. CD4+ cells were however identified in the lung and spleen of CD8+ recipients at d 13 making conclusions about the role of CD8+ cells in R. equi clearance impossible. By contrast, no significant increases in CD8+ lymphocytes were observed in the organs of CD4+ recipients. All mice developed suppurative bronchopneumonia but lesions were most severe in the CD4+ group. Immunohistochemistry and flow cytometry confirmed that CD4+ and CD8+ cells had migrated to the lungs of adoptively transferred mice. Serum antibody against R, equi was not detected by ELISA in the recipients. SCID/beige mice receiving CD4-CD8- cells were unable to clear R. equi. The study supports the suggestion that CD4+ cells have a central role in R. equi clearance in mice.