James Scott Miners

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.

Aβ-Degrading Enzymes: Potential for Treatment of Alzheimer Disease

There is increasing evidence that deficient clearance of &bgr;-amyloid (A&bgr;) contributes to its accumulation in late-onset Alzheimer disease (AD). Several A&bgr;-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce A&bgr; levels and protect against cognitive impairment in mouse models of AD. The activity of several A&bgr;-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of A&bgr;. The age- and disease-related changes in expression of more recently recognized A&bgr;-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of A&bgr;-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of A&bgr;-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other A&bgr;-degrading enzymes may offer advantages.

Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy

Neprilysin (NEP) degrades amyloid-&bgr; (A&bgr;) and is thought to contribute to its clearance from the brain. In Alzheimer disease (AD), downregulation of NEP has been suggested to contribute to the development of cerebral amyloid angiopathy (CAA). We examined the relationship among NEP, CAA, and APOE status in AD and elderly control cases. NEP was most abundant in the tunica media of cerebrocortical blood vessels and in pyramidal neurons. In homogenates of the frontal cortex, NEP protein levels were reduced in AD but not significantly; NEP enzymatic activity was significantly reduced in AD. Immunohistochemistry revealed a reduction of both vascular and parenchymal NEP. The loss of vessel-associated NEP in AD was inversely related to the severity of CAA, and analysis of cases with severe CAA showed that levels of vascular NEP were reduced to the same extent in A&bgr;-free and A&bgr;-laden vessels, strongly suggesting that the reduction in NEP is not simply secondary to CAA. Possession of APOE &egr;4 was associated with significantly lower levels of both parenchymal and vascular NEP. Colinearity of &egr;4 with the presence of moderate to severe CAA precluded assessment of the independence of this association from NEP levels. However, logistic regression analysis showed low NEP levels to be a significant independent predictor of moderate to severe 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.

Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity

Experimental reduction of neprilysin (NEP) or insulin-degrading enzyme (IDE) in vivo exacerbates &bgr;-amyloid accumulation in the brain. The level of these enzymes is reportedly reduced during aging and in postmortem brains of patients with sporadic Alzheimer disease (AD). To distinguish between primary decreases in NEP and IDE activity that might contribute to &bgr;-amyloid accumulation and decreases secondary to neurodegenerative changes in AD, we measured NEP and IDE levels by indirect sandwich ELISA and enzyme activities by immunocapture-based fluorogenic assays in postmortem frontal cortex from patients of different ages and at different pathological stages of AD, as indicated by Braak tangle stage. The ELISA measurements of neuron-specific enolase were used to adjust for neuronal loss. Both unadjusted and neuron-specific enolase-adjusted NEP levels and activity were significantly increased in AD and positively correlated with Braak stage but negatively with age in AD patients. Insulin-degrading enzyme activity was higher in AD than controls; this was significant after adjustment for neuron-specific enolase level; unadjusted IDE protein level was decreased in AD but not after adjustment. Our findings suggest that reduction in NEP and IDE activity is not the primary cause of &bgr;-amyloid accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

Insights into the pathogenesis and pathogenicity of cerebral amyloid angiopathy.

Amyloid-beta (Abeta) cerebral amyloid angiopathy (CAA) affects most Alzheimers disease (AD) patients and ~30% of otherwise-normal elderly people. APOE epsilon 4 is a major risk factor for CAA in AD. Neurons are probably the source of the vascular Abeta. CAA develops when Abeta is deposited in the vessel walls along or across which it normally passes into the CSF and bloodstream. Vascular deposition is facilitated by factors that increase Abeta40:Abeta42, impede perivascular passage of Abeta or raise its concentration. The levels of some Abeta-degrading enzymes are reduced in AD patients with CAA. However, angiotensin-converting enzyme activity is increased and may act via angiotensin II to increase transforming growth factor beta1, a potent inducer of ECM synthesis. CAA is a cause of intracerebral haemorrhage and cerebral ischaemic damage. In AD, neuritic degeneration is accentuated around Abeta-laden vessels. Rarely, CAA is associated with angiitis. The balance between parenchymal and cerebrovascular degradation of Abeta, and regulation of perivascular extracellular matrix production, are likely to be key determinants of Abeta distribution and pathogenicity within the brain.

Oligomeric Aβ in Alzheimer's Disease: Relationship to Plaque and Tangle Pathology, APOE Genotype and Cerebral Amyloid Angiopathy

Despite accumulating evidence of a central role for oligomeric amyloid β (Aβ) in the pathogenesis of Alzheimers Disease (AD), there is scant information on the relationship between the levels and distribution of oligomeric Aβ and those of other neurodegenerative abnormalities in AD. In the present study, we have found oligomeric Aβ to be associated with both diffuse and neuritic plaques (mostly co‐localized with Aβ1–42) and with cerebrovascular deposits of Aβ in paraffin sections of formalin‐fixed human brain tissue. The amount of oligomeric Aβ that was labeled in the sections correlated with total Aβ plaque load, but not phospho‐tau load, cerebral amyloid angiopathy (CAA) severity or APOE genotype. Although soluble, oligomeric and insoluble Aβ levels were all significantly increased in AD brain homogenates, case‐to‐case variation and overlap between AD and controls were considerable. Over the age‐range studied (43–98 years), the levels of soluble Aβ, oligomeric Aβ42, oligomeric Aβ40 and insoluble Aβ did not vary significantly with age. Oligomeric Aβ1–42 and insoluble Aβ levels were significantly higher in women. Overall, the level of insoluble Aβ, but neither oligomeric nor soluble Aβ, was associated with Braak stage, CAA severity and APOEε4 frequency, raising questions as to the role of soluble and oligomeric Aβ in the progression of AD.

Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimers disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.

Neprilysin Protects against Cerebral Amyloid Angiopathy and Aβ‐Induced Degeneration of Cerebrovascular Smooth Muscle Cells

Neprilysin (NEP), which degrades amyloid‐β (Aβ), is expressed by neurons and cerebrovascular smooth muscle cells (CVSMCs). NEP immunolabeling is reduced within cerebral blood vessels of Alzheimers disease (AD) patients with cerebral amyloid angiopathy (CAA). We have now measured NEP enzyme activity in leptomeningeal and purified cerebral cortical blood vessel preparations from control and AD patients with and without CAA. Measurements were adjusted for smooth muscle actin (SMA) to control for variations in CVSMC content. NEP activity was reduced in CAA, in both controls and AD. In leptomeningeal vessels, NEP activity was related to APOE genotype, being highest in ε2‐positive and lowest in ε4‐positive brains. To assess the role of NEP in protecting CVSMCs from Aβ toxicity, we measured cell death in primary human adult CVSMCs exposed to Aβ1‐40, Aβ1‐42 or Aβ1‐40(Dutch variant). Aβ1‐42 was most cytotoxic to CVSMCs. Aβ1‐42‐mediated cell death was increased following siRNA‐mediated knockdown or thiorphan‐mediated inhibition of NEP activity; conversely Aβ1‐42‐mediated cytotoxicity was reduced by the addition of somatostatin and NEP over‐expression following transfection with NEP cDNA. Our findings suggest that NEP protects CVSMCs from Aβ toxicity and protects cerebral blood vessels from the development and complications of CAA.

Accumulation of Insoluble Amyloid-β in Down's Syndrome is Associated with Increased BACE-1 and Neprilysin Activities

We previously reported age- and Alzheimers disease (AD)-related increases in the activities of β-secretase (BACE-1) and Aβ-degrading enzymes including neprilysin (NEP) and angiotensin-converting enzyme (ACE) in the frontal cortex. We suggested that these increases were secondary to the accumulation of insoluble amyloid-β (Aβ) and a decline in soluble Aβ. We have further tested this hypothesis by examination of frontal cortex obtained postmortem from individuals with Downs syndrome (DS), in whom AD-like neuropathological changes occur in association with early-onset dementia. We measured total soluble and insoluble (guanidine-extractable) Aβ, BACE-1 activity, and the concentrations and activities of NEP and ACE in two independent DS cohorts: an initial, Bristol cohort (9 DS cases, 8 controls matched for age-at-death) and a validation Newcastle cohort (20 DS, 18 controls with a wider spectrum of age-at-death). In both cohorts the level of insoluble (but not soluble) Aβ was significantly higher in DS than controls and was comparable to previously measured levels in AD. NEP protein concentration and activity were significantly increased in DS; a trend towards increased BACE-1 activity was observed in DS but did not reach statistical significance. Both NEP and BACE-1 correlated with the level of insoluble Aβ. The concentration of ACE in DS was elevated in the pilot cohort only and ACE activity was unchanged. These findings provide strong support that BACE-1 and NEP activities, but not ACE, increase in response to the accumulation of insoluble Aβ within the brain.