Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor

Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.     

Conformational differences between two amyloid β oligomers of similar size and dissimilar toxicity

Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid β (Aβ42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aβ42 spontaneously forms prefibrillar oligomers at Aβ concentrations below 30 μm in the absence of agitation, whereas higher Aβ concentrations lead to rapid formation of fibrils. Interestingly, Aβ prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aβ prefibrillar oligomers. Strikingly, this alternative Aβ oligomer is non-toxic to mammalian cells relative to Aβ monomer. We find that two hydrophobic peptide segments within Aβ (residues 16-22 and 30-42) are more solvent-exposed in the more toxic Aβ oligomer. The less toxic oligomer is devoid of β-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aβ oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.     

Differences in resolution of mwr-containing plasmid dimers mediated by the Klebsiella pneumoniae and Escherichia coli XerC recombinases: potential implications in dissemination of antibiotic resistance genes

Xer-mediated dimer resolution at the mwr site of the multiresistance plasmid pJHCMW1 is osmoregulated in Escherichia coli containing either the Escherichia coli Xer recombination machinery or Xer recombination elements from K. pneumoniae. In the presence of K. pneumoniae XerC (XerC(Kp)), the efficiency of recombination is lower than that in the presence of the E. coli XerC (XerC(Ec)) and the level of dimer resolution is insufficient to stabilize the plasmid, even at low osmolarity. This lower efficiency of recombination at mwr is observed in the presence of E. coli or K. pneumoniae XerD proteins. Mutagenesis experiments identified a region near the N terminus of XerC(Kp) responsible for the lower level of recombination catalyzed by XerC(Kp) at mwr. This region encompasses the second half of the predicted alpha-helix B and the beginning of the predicted alpha-helix C. The efficiencies of recombination at other sites such as dif or cer in the presence of XerC(Kp) or XerC(Ec) are comparable. Therefore, XerC(Kp) is an active recombinase whose action is impaired on the mwr recombination site. This characteristic may result in restriction of the host range of plasmids carrying this site, a phenomenon that may have important implications in the dissemination of antibiotic resistance genes.     

Amyloid β-Protein Induces Its Own Production in Cultured Degenerating Cerebrovascular Smooth Muscle Cells

Abstract: The progression of Alzheimer's disease and related disorders involves amyloid β-protein (Aβ) deposition and pathologic changes in the parenchyma as well as cerebral blood vessels. The cerebrovascular Aβ deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall, which have been shown to express the Aβ precursor (AβPP) and Aβ. Here, we show that Aβ_1–42, an abundant cerebrovascular form of Aβ, causes cellular degeneration in cultured human cerebrovascular smooth muscle cells. This stress response is accompanied by a striking increase in the levels of cellular AβPP and soluble Aβ peptide produced in these degenerating cells. These data provide the first experimental evidence that Aβ can potentially contribute to the onset and progression of the cerebrovascular pathology. The present findings suggest that this mechanism may involve a molecular cascade with a novel product-precursor relationship that results in the adverse production and subsequent accumulation of Aβ.     

Early-onset Formation of Parenchymal Plaque Amyloid Abrogates Cerebral Microvascular Amyloid Accumulation in Transgenic Mice

The fibrillar assembly and deposition of amyloid β (Aβ) protein, a key pathology of Alzheimer disease, can occur in the form of parenchymal amyloid plaques and cerebral amyloid angiopathy (CAA). Familial forms of CAA exist in the absence of appreciable parenchymal amyloid pathology. The molecular interplay between parenchymal amyloid plaques and CAA is unclear. Here we investigated how early-onset parenchymal amyloid plaques impact the development of microvascular amyloid in transgenic mice. Tg-5xFAD mice, which produce non-mutated human Aβ and develop early-onset parenchymal amyloid plaques, were bred to Tg-SwDI mice, which produce familial CAA mutant human Aβ and develop cerebral microvascular amyloid. The bigenic mice presented with an elevated accumulation of Aβ and fibrillar amyloid in the brain compared with either single transgenic line. Tg-SwDI/Tg-5xFAD mice were devoid of microvascular amyloid, the prominent pathology of Tg-SwDI mice, but exhibited larger parenchymal amyloid plaques compared with Tg-5xFAD mice. The larger parenchymal amyloid deposits were associated with a higher loss of cortical neurons and elevated activated microglia in the bigenic Tg-SwDI/Tg-5xFAD mice. The periphery of parenchymal amyloid plaques was largely composed of CAA mutant Aβ. Non-mutated Aβ fibril seeds promoted CAA mutant Aβ fibril formation in vitro. Further, intrahippocampal administration of biotin-labeled CAA mutant Aβ peptide accumulated on and adjacent to pre-existing parenchymal amyloid plaques in Tg-5xFAD mice. These findings indicate that early-onset parenchymal amyloid plaques can serve as a scaffold to capture CAA mutant Aβ peptides and prevent their accumulation in cerebral microvessels.     

Localization of a fibrillar amyloid beta-protein binding domain on its precursor

Deposition of fibrillar amyloid-beta protein (Abeta) in senile plaques and in the walls of cerebral blood vessels is a key pathological feature of Alzheimer's disease and certain related disorders. Fibrillar Abeta deposition is intimately associated with neuronal and cerebrovascular cell death both in vivo and in vitro. Similarly, accumulation of the Abeta protein precursor (AbetaPP) is also observed at sites of fibrillar Abeta deposition. Recently, we reported that fibrillar Abeta, but not unassembled Abeta, promotes the specific binding of AbetaPP through its cysteine-rich, amino-terminal region (Melchor, J. P., and Van Nostrand, W. E. (2000) J. Biol. Chem. 275, 9782-9791). In the present study we sought to determine the precise site on AbetaPP that facilitates its binding to fibrillar Abeta. A series of synthesized overlapping peptides spanning the cysteine-rich, amino-terminal region of AbetaPP were used as competitors for AbetaPP binding to fibrillar Abeta. A peptide spanning residues 105-119 of AbetaPP competitively inhibited AbetaPP binding to fibrillar Abeta in a solid-phase binding assay and on the surface of cultured human cerebrovascular smooth muscle cells. Alanine-scanning mutagenesis of residues 105-117 within glutathione S-transferase (GST)-AbetaPP-(18-119) revealed that His(110), Val(112), and Ile(113) are key residues that facilitate AbetaPP binding to fibrillar Abeta. These specific residues belong to a common beta-strand within this region of AbetaPP. Wild-type GST-AbetaPP-(18-119) protected cultured human cerebrovascular smooth muscle cells from Abeta-induced toxicity whereas H110A mutant GST-AbetaPP-(18-119) did not. Wild-type GST-AbetaPP-(18-119) bound to different isoforms of fibrillar Abeta and fibrillar amylin peptides whereas H110A mutant and I113A mutant GST-AbetaPP-(18-119) were substantially less efficient binding to each fibrillar peptide. We conclude that His(110), Val(112), and Ile(113), residing in a common beta-strand region within AbetaPP-(18-119), comprise a domain that mediates the binding of AbetaPP to fibrillar peptides.     

Amyloid beta-protein stimulates the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in human cerebrovascular smooth muscle cells

The accumulation of fibrillar amyloid-beta protein (A beta) in cerebral blood vessels, a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and certain related disorders and is intimately associated with cerebrovascular cell death both in vivo and in vitro. Moreover, severe CAA leads to loss of vessel wall integrity and cerebral hemorrhage. Although the basis for these latter pathological consequences in CAA remains unresolved alterations in local proteolytic mechanisms may be involved. Here we show that pathogenic forms of A beta stimulate the expression of plasminogen activator activity in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of CAA. RNase protection assays and plasminogen zymography showed that urokinase-type plasminogen activator (uPA) was responsible for this activity. There was preferential accumulation of uPA on the HCSM cell surface that was mediated through a concomitant increase in expression of the uPA receptor. In the presence of plasminogen there was robust degradation of A beta that was added to the HCSM cells resulting in restoration of cell viability. This suggests that increased expression of uPA may initially serve as a protective mechanism leading to localized degradation and clearance of the pathogenic stimulus A beta. On the other hand, chronic expression of uPA and plasminogen activation led to a profound loss of HCSM cell attachment. This suggests that a similar prolonged effect in vivo in the cerebral vessel wall may contribute to loss of integrity and cerebral hemorrhage in CAA.     

N-terminal Domain of Myelin Basic Protein Inhibits Amyloid β-Protein Fibril Assembly

Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952–9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720–4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.     

Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aβ40 fibrils

The accumulation of fibrillar amyloid-β (Aβ) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer''s disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aβ mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aβ fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel β-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aβ fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aβ fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aβ40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel β-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register β-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aβ fibrils in the human brain.     

West Nile Virus Retinopathy and Associations with Long Term Neurological and Neurocognitive Sequelae

West Nile virus (WNV) has emerged as an important vector-borne pathogen in North America, with more than 3 million estimated to have been infected. Retinopathy from WNV infection has been previously reported in acute cases, though those prior reports did not evaluate the risk of retinopathy based on clinical severity of neurologic disease. The purpose of this cross-sectional study was to perform comprehensive ophthalmological and neurological examinations on 111 patients with a history of West Nile virus infection and describe the ocular manifestations. Out of 111 patients, 27 (24%) had evidence for West Nile virus associated retinopathy (WNVR); this observation was higher (49%) in those patients who initially presented with encephalitis. Individuals with WNVR had more frequent involvement of the macula and peripheral involvement compared to those patients without WNVR (p<0.05). WNVR was also associated with a greater likelihood of abnormal reflexes on neurological exam, poorer learning, greater dependence in activities of daily living, and lower quality of life (p<0.05). WNVR was seen more frequently in elderly patients (age > 60 years), and was associated with higher rates of diabetes mellitus and a history of encephalitis (p<0.05). A multivariable logistic regression revealed that only a history of encephalitis was independently associated with WNVR [Adjusted Odds Ratio = 4.9 (1.8–13.2); p = 0.001]. Our study found that WNVR occurs in one fourth of patients with a history of WNV infection and is more frequently observed in those with apparent severe neurological sequelae (e.g., encephalitis). The clinical relevance of WNVR was supported by its associations with dependence in activities of daily living and lower quality of life. This unique evaluation of WNV patients included fundoscopic examinations and their associations with neurologic impairment. Our findings can be used during ophthalmological consultation for the evaluation, treatment and rehabilitation phases of care for WNV patients.