Theresa A. Davies

Toxicity of various amyloid β peptide species in cultured human blood–brain barrier endothelial cells: Increased toxicity of Dutch‐type mutant

The amyloid β peptide (Aβ) is the major component of the neuritic and cerebrovascular amyloid plaques that are one of the characteristic features of Alzheimers disease (AD). This peptide has been shown to be toxic to several relevant cell types, including neurons, cerebrovascular smooth muscle cells, and endothelial cells. We have studied the toxic effects of both soluble and aggregated species of Aβ1–40 and the mutation Aβ1–40Glu→Gln22, which is the major species deposited in the cerebrovascular blood vessels of victims of hereditary cerebral hemorrhage with amyloidosis, Dutch type. We find that aggregates of both peptides, as well as of Aβ1–42 and Aβ25–35, are toxic to cultured human cerebrovascular endothelial cells (hBEC) obtained from the brain of a victim of AD (at doses lower than those that are toxic to CNS neurons or leptomeningeal smooth muscle cells). Soluble Aβ1–40 Gln22 is equally toxic to hBEC, whereas wild‐type Aβ1–40 is toxic only at higher doses. This toxicity is seen at the lowest dose of Aβ1–40 Gln 22 used, 20 nM. The soluble Aβ1–40Gln22 aggregates on the surface of the cells, in contrast to Aβ1–40, and its toxicity can be blocked both by an inhibitor of free radical formation and by Congo red, which inhibits amyloid fibril formation. We discuss the possibility that the enhanced toxicity of Aβ1–40Gln22 is mediated by a Aβ receptor on the endothelial cells. J. Neurosci. Res. 60:804–810, 2000.

p amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium: usage of a novel blood brain barrier endothelial cell model system

Amyloid precursor protein (AβPP) processing results in generation of amyloid β peptide (Aβ) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimers disease (AD). Evidence that the vascular deposits derive in part from AβPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken antiplatelet drugs. Thus, determination ofwhether platelet AβPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface trans-membrane-bound AβPP (mAβPP) than control platelets. We report here that this mAβPP can be cleaved to Aβ-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mAβPP, cleave fluorescein-tagged surface proteins, including mAβPP, resulting in passage to the BEC layer. The data confirm that BEC contribute to processing of platelet-derived mAβPP and show that the processing yields Aβ con-tainingfragments which could potentially contribute to cerebrovascular Aβ deposition.

Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimers disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Platelets and DAMI megakaryocytes possess β-secretase-like activity

Abstract We report here the discovery of two novel human platelet and megakaryocytic DAMI cell enzymes that have β-secretase-like activity. These activities could potentially effect cleavage of the amyloid precursor protein (APP) at the β-amyloid peptide N-terminus, by an EC 3.4.24.15-like metalloprotease, and the N terminus-1 position, by a serine protease. Thus both enzymes may generate the amyloidogenic β-peptide. Studies of intact and Triton X-100-lysed DAMI cells, as well as intact versus subcellular fractions of platelets, demonstrate the presence of these proteolytic activities. The resting platelet has (1) a surface serine protease, demonstrated by its ability to cleave a β-secretase substrate and by its inhibitor sensitivity; and (2) a metalloprotease, recognized by an antibody to EC 3.4.24.15, which resides intracellularly in the α-granule membrane, is translocated to the surface on activation, and shows β-secretase-like activity by cleaving the same substrate. This metalloprotease can also cleave recombinant APP to a potentially amyloidogenic fragment. Surface metalloprotease was identified in DAMI cells by flow cytometry and Western blotting with a specific anti-EC 3.4.24.15 monoclonal antibody, while activity was identified by using two β-secretase substrates. This article is the first to document two previously unknown endoproteinases with β-secretase-like activity in platelets and DAMI cells. These proteases are capable of effecting cleavage of APP and could therefore contribute to Aβ deposition in the cerebrovasculature. (J Lab Clin Med 1999;133:507-15)

Brain endothelial cell enzymes cleave platelet-retained amyloid precursor protein

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimers disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

Platelets from patients with Alzheimer's disease or other dementias exhibit disease-specific and apolipoprotein E correlatable defects

Platelets carry over 95% of the circulating Alzheimers β-amyloid precursor protein (AβPP), and release soluble and hydrophobic proteolytic fragments of AβPP upon activation. These cells may be the source of cerebrovascular amyloid peptides, a part of Alzheimers disease (AD) pathology. Our previous studies showed that platelets from patients with advanced AD exhibit both signal transduction (hyperacidification) and AβPP processing defects. Here, we show further that a similar hyperacidification also exists in patients with Picks disease (a dementia with AD-like symptoms but a different amyloid pathology) or Down syndrome (trisomy and hence overproduction of AβPP), while the AβPP processing defect and consequent AβPP retention on the membrane is absent and is thus likely to be AD-specific. The hyperacidiftcation defect correlates with all three dementias and with the presence of apolipoprotein E4 which has been implicated as a risk factorial-AD.

Sequential sodium-proton exchange in thrombin-induced human platelets

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.

Cytosolic calcium determination: a fluorometric technique

After human platelet and leucocyte isolation, and Indo-1 acetoxymethyl ester loading, the fluorometric technique of flow cytometric measurments has been used to analyse changes in cytosolic calcium. Measurments in cells suspensions and in individual cells.

Identification and Characterization of Functional Secretory Cells: Advantages of Multiparameter Flow Cytometry Kinetics

Publisher Summary Flow cytometers were initially designed to permit identification of cells by means of their individual, highly specific, and therefore uniquely identifying surface proteins that served as antigens to well-characterized monoclonal or polyclonal antibodies. These, in turn, were detected either by their own fluorescent markers or by appropriately labeled second antibodies. The usual labels remain fluorescein, rhodamine, and phycoerythrin derivatives. The light scattering profiles of certain cells (e.g., those of the circulating blood), were also found to be fairly consistent and specific, thus permitting some cell characterization based on differences in cell size, with right angle and forward light-scatter profiles pragmatically related to the granularity and the size of the cell, respectively. All of these applications utilized equilibrated cells, whether fixed, permeabilized, or native. More recently, the ability to introduce samples and obtain data rapidly, as well as improvements in instrumentation, in fluorescent probe technology, and in antibody development, have permitted flow cytometry to advance from its initial application, and have enabled flow cytometers to be used for kinetic measurements and therefore to follow the responses of living and fully functional cells. Because some are equipped to measure up to six parameters simultaneously using four photomultipliers, several parameters of cell function can be correlated, for any given cell, with occupancy of its receptors.

β amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium : Usage of a novel blood brain barrier endothelial cell model system

Amyloid precursor protein (A betaPP) processing results in generation of amyloid beta peptide (A beta) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimers disease (AD). Evidence that the vascular deposits derive in part from A betaPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken anti-platelet drugs. Thus, determination of whether platelet A betaPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface transmembrane-bound A betaPP (mA betaPP) than control platelets. We report here that this mA betaPP can be cleaved to A beta-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mA betaPP, cleave fluorescein-tagged surface proteins, including mA betaPP, resulting in passage to the BEC layer The data confirm that BEC contribute to processing of platelet-derived mA betaPP and show that the processing yields A beta containing fragments which could potentially contribute to cerebrovascular A beta deposition.