William E. Van Nostrand

Charge Alterations of E22 Enhance the Pathogenic Properties of the Amyloid β-Protein

Abstract: Cerebral amyloid angiopathy (CAA) due to amyloid β‐protein (Aβ) is a key pathological feature of patients with Alzheimers disease and hereditary cerebral hemorrhage with amyloidosis, Dutch‐type (HCHWA‐D). The CAA in these disorders is characterized by deposition of Aβ in the smooth muscle cells within the cerebral vessel wall. Recently, a new mutation in Aβ, E22K, was identified in several Italian families that, like HCHWA‐D, is associated with CAA and hemorrhagic stroke. These two similar disorders, stemming from amino acid substitutions at position 22 of Aβ, implicate the importance of this site in the pathology of HCHWA. Previously we showed that HCHWA‐D Aβ1‐40 containing the E22Q substitution induces robust pathologic responses in cultured human cerebrovascular smooth muscle cells (HCSM cells), including highly elevated levels of cell‐associated Aβ precursor (AβPP) and cell death. In the present study, a series of E22 mutant Aβ1‐40 peptides were synthesized, and their pathogenic properties toward cultured HCSM cells were evaluated. Quantitative fluorescence analyses showed that mutant Aβ1‐40 peptides either containing a loss of charge (E22Q and E22A) or a change of charge (E22K) bind to the surface of HCSM cells and form amyloid fibrils. Similarly, this same group of E22 mutant Aβ1‐40 peptides caused enhanced pathologic responses in HCSM cells. In contrast, wild‐type E22 or the charge‐preserving E22D Aβ1‐40 peptides were devoid of any of these pathogenic properties. These data suggest that a change or loss of charge at position 22 of Aβ enhances the pathogenic effects of the peptide toward HCSM cells and may contribute to the pathogenesis of the phenotypically related HCHWA disorders.

Pathologic Amyloid β‐Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells

Abstract: Cerebrovascular amyloid β‐protein (Aβ) deposition is a key pathological feature of Alzheimers disease and hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D). Aβ1–40 containing the E22Q HCHWA‐D mutation, but not wild‐type Aβ1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µM neither HCHWA‐D Aβ1–40 nor wild‐type Aβ1–40 appreciably assembles into β‐pleated sheet‐containing fibrils in solution over a 6‐day incubation period. In contrast, at the same concentrations, HCHWA‐D Aβ1–40, but not wild‐type Aβ1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA‐D Aβ1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA‐D Aβ1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.

Amyloid-beta-induced degeneration of human brain pericytes is dependent on the apolipoprotein E genotype.

Abstract: Amyloid‐β (Aβ) deposition in cerebral vessels (cerebral amyloid angiopathy, CAA) is accompanied by degeneration of vascular cells, including pericytes and smooth muscle cells. Previous studies indicated that specific Aβ protein isoforms are toxic for cultured human brain pericytes and smooth muscle cells. In particular, Aβ1–40 carrying the E22Q mutation, as in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA‐D), is toxic. We investigated the effects of the Aβ‐binding protein apolipoprotein E (ApoE) on the toxicity of Aβ for cultured human brain pericytes. We compared the toxicity of HCHWA‐D Aβ1–40 for pericyte cultures with different ApoE genotypes, studied the accumulation of Aβ and ApoE in these different cell cultures, and investigated the effects of exogenous ApoE. Pericyte cultures with an ApoE ɛ2/ɛ3 genotype were more resistant to HCHWA‐D Aβ1–40 treatment than cultures with a ɛ3/ɛ3 or ɛ3/ɛ4 genotype. Cell death was highest in cultures homozygous for ApoE ɛ4. The extent to which both Aβ and ApoE accumulated at the cell surface was parallel to the degree of toxicity. The addition of purified ApoE resulted in a decrease in cell death. These data suggest that ApoE4 may direct Aβ more efficiently than other ApoE isoforms into a pathological interaction with the HBP cell surface. The results of this study are in line with the observations that inheritance of the ApoE ɛ4 allele increases the risk of developing Alzheimers disease, and that the ApoE ɛ2 allele has a relatively protective effect.

Pathogenic amyloid β-protein induces apoptosis in cultured human cerebrovascular smooth muscle cells

The amyloid pprotein (Aβ) pathologically accumulates in cerebral vascular and senile plaque deposits in the brains of patients with Alzheimers disease (AD) and related dis-A, orders including hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). The cerebrovascular deposits are accompanied by degeneration and eventual loss of smooth muscle cells in cerebral vessel wall. Similarly, we have shown that pathogenic forms of Aβ cause cell death in cultured human Cerebrovascular smooth muscle (HCSM) cells in vitro. Here we show that pathogenic Aβ induces a number of structural changes in HCSM cells including shrinkage of cell bodies, retraction of processes, disruption of the intracellular act in network, and nuclear condensation and fragmentation. These changes were accompanied by a number of biochemical alterations in the cells shown by in situ end labeling of nuclear DNA, proteolytic breakdown of smooth muscle cell a acting, and proteolytic activation of the proteinase caspase 3. Together, th...

Cerebrovascular Smooth Muscle Cell Surface Fibrillar Aβ: Alteration of the Proteolytic Environment in the Cerebral Vessel Wall

Abstract: Cerebrovascular deposition of the amyloid b‐protein (Aβ) is a common pathologic event in patients with Alzheimers disease (AD) and certain related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch‐type (HCHWA‐D). Aβ deposition occurs primarily in the medial layer of the cerebral vessel wall in an assembled fibrillar state. These deposits are associated with several pathological responses including degeneration of the smooth muscle cells in the cerebral vessel wall. Severe cases of cerebrovascular Aβ deposition are also accompanied by loss of vessel wall integrity and hemorrhagic stroke. Although the reasons for this pathological consequence are unclear, altered proteolytic mechanisms within the cerebral vessel wall may be involved. Recent studies from our laboratory have shown that cell‐surface assembly of Aβ into fibrillar structures causes cellular degeneration via an apoptotic pathway and creates an altered proteolytic microenvironment on the cell surface of human cerebrovascular smooth muscle cells (HCSM cells). For example, HCSM cell‐surface Aβ fibrils serve as a site for tight binding of cell‐secreted amyloid β‐precursor protein (AβPP). Since AβPP is a potent inhibitor of key proteinases of coagulation cascade, its enhanced localization on the Aβ fibrils would provide an strong anticoagulant environment. In addition, HCSM cell‐surface Aβ fibrils are potent stimulators of tissue plasminogen activator (tPA) creating a profibrinolytic milieu. Our findings indicate that Aβ fibril assembly on the HCSM cell surface causes cellular degeneration and results in both a strong anticoagulant and fibrinolytic environment. Together, these altered proteolytic events could create a setting that is conducive to loss of vessel wall integrity and hemorrhagic stroke.

Pathogenic Effects of Cerebral Amyloid Angiopathy Mutations in the Amyloid β-Protein Precursor

Abstract: Cerebral amyloid β‐protein angiopathy (CAA) is a key pathological feature of patients with Alzheimers disease and certain related disorders. Several mutations have been identified within the Aβ region of the Aβ protein precursor (AβPP) gene that appear to enhance the severity of CAA. A new mutation has been identified within the Aβ region (D23N) of AβPP that is associated with severe CAA in an Iowa kindred. Recently, we showed that E22Q Dutch, D23N Iowa, and E22Q/D23N Dutch/Iowa double‐mutant Aβ40 peptides rapidly assemble in solution to form fibrils compared to wild‐type Aβ40. Similarly, the E22Q Dutch and D23N Iowa Aβ40 peptides were found to induce robust pathologic responses in cultured human cerebrovascular smooth muscle (HCSM) cells, including elevated levels of cell‐associated AβPP, proteolytic breakdown of actin, and cell death. Double‐mutant E22Q/D23N Dutch/Iowa Aβ40 was more potent than either single‐mutant form of Aβ in causing pathologic responses in HCSM cells. These in vitro data suggested that the E22Q Dutch and D23N Iowa substitutions promote fibrillogenesis and the pathogenicity of Aβ towards HCSM cells. Moreover, the presence of both CAA substitutions in the same Aβ peptide further enhances the fibrillogenic and pathogenic properties of Aβ. We also have generated transgenic mouse models to examine the effects of single and double CAA mutations in AβPP in vivo. Preliminary analysis of transgenic mouse brains indicates that expression of double‐mutant E22Q/D23N Dutch/Iowa AβPP leads to robust deposition of Aβ in a vascular‐weighted manner.

Identification of a novel serine protease-like molecule in human brain.

Proteolysis of the amyloid beta protein precursor (APP) is a key event in the development of Alzheimers disease. In our search for proteases that can cleave APP and liberate the amino terminus of the amyloidogenic beta protein, we characterized a calcium-dependent serine protease (CASP) which is present in reactive astrocytes and cross-reacts with anti-cathepsin G antibodies. We wanted to take advantage of this cross-reactivity to clone the cDNA of CASP and eventually evaluate its tissue distribution. Screening of two human fetal brain cDNA libraries with anti-cathepsin G antibodies led to the identification of a cDNA coding for a novel protein whose only homology to known proteins is to the active site of trypsin-type serine proteases. We called this protein the novel serine protease (NSP). NSP exists in at least three differentially spliced forms, one of which is expressed predominantly in brain and testis. Immunohistochemistry and immunoprecipitation with antibodies generated against NSP show that it is expressed and secreted by a variety of cells and that, in brain, it is found primarily in cerebrovascular smooth muscle cells and reactive astrocytes.

Degeneration of Human Cerebrovascular Smooth Muscle Cells and Pericytes Caused by Amyloid β Protein

The deposition of amyloid β protein in the cerebral vasculature (also known as cerebral amyloid angiopathy, CAA, or congophilic angiopathy) is one of the neuropathological hallmarks of Alzheimer’s disease and several related disorders, such as hereditary cerebral hemorrhage with amyloidosis of the Dutch type. CAA can also occur independently of Alzheimer’s disease and is common in the brain of elederly individuals. When this condition develops in an advanced stage, it may be associated with cerebral hemorrhages and stroke. CAA-related brain hemorrhages often occur in association with brain microvascular alterations that include fibrinoid necrosis, microaneurysm formation and smooth muscle cell and pericyte death. In this review we summarize the relationship between amyloid β peptides and degeneration of smooth muscle cells and pericytes that have been studied in vitro. In particular, the effects of specific Aβ isoforms and the role of peptide fibril assembly in the induction of cellular degeneration and in increased cell-associated amyloid precursor protein has been extensively investigated. Evidence will be presented to demonstrate a differential response to different Aβ assembly states that mediate the toxic effect of the peptide on cerebrovascular cells on the one hand, and neurons on the other. These data suggest that there may be different recognition and/or pathogenic mechanisms for cells in the cerebrovascular compartment compared to neuronal cells.

Amyloid

The amyloid β-protein (Aβ) which accumulates in cerebral vascular and senile plaque deposits in the brains of patients with Alzheimers disease (AD) is proteolytically derived from a larger precursor protein, the amyloid β-protein precursor (AβPP). AβPP is a member of a multigene family which includes amyloid precursor-like protein 2 (APLP2). Recently, we showed that Aβ1-42, but not the shorter Aβ1-40 induces cellular levels of AβPP in degenerating cultured human cerebrovascular smooth muscle (HCSM) cells. Here we report that Aβ1-42 stimulates a parallel increase in the cellular levels of APLP2 in cultured HCSM cells. in contrast, the levels of smooth muscle cell α-actin or total cellular protein did not appreciably change. These findings suggest a common regulatory mechanism for increased levels of HCSM cellular AJPP and APLP2 in response to pathologic Aβ1-42 induced stress.

sZ-protein stimulates parallel increases in cellular levels of its precursor and amyloid precursor-like protein 2 (APLP2) in human cerebrovascular smooth muscle cells

Abstract Hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D), caused by a mutation at codon 693 in the amyloid β precursor protein (βPP) gene, is clinically characterised by haemorrhagic strokes and dementia. The secreted forms βPP751 and βPP770 are identical to protease nexin II (PNII), which is a potent inhibitor of intrinsic blood coagulation factor XIa in vitro. We investigated the concentration of complexes between factor XIa and βPP in vivo, to search for a possible cause of the haemorrhagic strokes in HCHWA-D. In this prospectively designed study, first an enzyme-linked immunosorbent assay (ELISA) was performed with dilutions of the factor XIa-βPP complexes prepared from platelets as a standard curve. By means of this ELISA, the concentration of factor XIa-βPP complexes was measured in plasma samples. Second, plasma of 23 HCHWA-D patients and 23 healthy controls was collected, and the concentration of complexes was measured with this method. The mean concentration of factor XIa-βPP complexes in plasma of HCHWA-D patients (mean 13.73 U/ ml; SD 4.78) was significantly (P = 0.05) higher than the concentration in plasma of healthy controls (mean 11.37; SD 2.77). The differences, however, were small and there was a major overlap between the concentrations in patients and controls. In HCHWA-D mutation carriers, the concentration of factor XIa-βPP complexes was not related to age, and there was no difference between presymptomatic and symptomatic mutation carriers. From this study, it can be concluded that βPP forms complexes with factor XIa in vivo both in HCHWA-D patients and in normal controls. The concentration in HCHWA-D patients was higher. This is probably the result of diffusion of complexes from the cerebral circulation into plasma. Elevated βPP in cerebrovascular amyloid deposits is possibly a local factor contributing to the development of haemorrhagic strokes.