Reduced Levels of Cerebrospinal Fluid/Plasma Aß40 as an Early Biomarker for Cerebral Amyloid Angiopathy in RTg-DI Rats.

The accumulation of fibrillar amyloid ß-protein (Aß) in blood vessels of the brain, the condition known as cerebral amyloid angiopathy (CAA), is a common small vessel disease that promotes cognitive impairment and is strongly associated with Alzheimer's disease. Presently, the clinical diagnosis of this condition relies on neuroimaging markers largely associated with cerebral macro/microbleeds. However, these are markers of late-stage disease detected after extensive cerebral vascular amyloid accumulation has become chronic. Recently, we generated a novel transgenic rat model of CAA (rTg-DI) that recapitulates multiple aspects of human CAA disease with the progressive accumulation of cerebral vascular amyloid, largely composed of Aß40, and the consistent emergence of subsequent microbleeds. Here, we investigated the levels of Aß40 in the cerebrospinal fluid (CSF) and plasma of rTg-DI rats as CAA progressed from inception to late stage disease. The levels of Aß40 in CSF and plasma precipitously dropped at the early onset of CAA accumulation at three months of age and continued to decrease with the progression of disease. Notably, the reduction in CSF/plasma Aß40 levels preceded the emergence of cerebral microbleeds, which first occurred at about six months of age, as detected by in vivo magnetic resonance imaging and histological staining of brain tissue. These findings support the concept that reduced CSF/plasma levels of Aß40 could serve as a biomarker for early stage CAA disease prior to the onset of cerebral microbleeds for future therapeutic intervention.

A Novel Transgenic Rat Model of Robust Cerebral Microvascular Amyloid with Prominent Vasculopathy.

Accumulation of fibrillar amyloid ß protein in blood vessels of the brain, a condition known as cerebral amyloid angiopathy (CAA), is a common pathology of elderly individuals, a prominent comorbidity of Alzheimer disease, and a driver of vascular cognitive impairment and dementia. Although several transgenic mouse strains have been generated that develop varying levels of CAA, consistent models of associated cerebral microhemorrhage and vasculopathy observed clinically have been lacking. Reliable preclinical animal models of CAA and microhemorrhage are needed to investigate the molecular pathogenesis of this condition. Herein, we describe the generation and characterization of a novel transgenic rat (rTg-DI) that produces low levels of human familial CAA Dutch/Iowa E22Q/D23N mutant amyloid ß protein in brain and faithfully recapitulates many of the pathologic aspects of human small-vessel CAA. rTg-DI rats exhibit early-onset and progressive accumulation of cerebral microvascular fibrillar amyloid accompanied by early-onset and sustained behavioral deficits. Comparable to CAA in humans, the cerebral microvascular amyloid in rTg-DI rats causes capillary structural alterations, promotes prominent perivascular neuroinflammation, and produces consistent, robust microhemorrhages and small-vessel occlusions that are readily detected by magnetic resonance imaging. The rTg-DI rats provide a new model to investigate the pathogenesis of small-vessel CAA and microhemorrhages, to develop effective biomarkers for this condition and to test therapeutic interventions.

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid ß-protein precursor abolishes its anti-thrombotic properties in vivo.

INTRODUCTION: Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid ß-protein precursor (AßPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AßPP are abundant in brain. Regions of AßPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AßPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. METHODS: To determine the extent of KPI function of AßPP in regulating cerebral thrombosis we generated a recombinant reactive center KPIR13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AßPP knock out and AßPP/KPIR13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. RESULTS: Recombinant mutant KPIR13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AßPP/KPIR13I mutant mice were similarly deficient as AßPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. CONCLUSIONS: We demonstrate that the anti-thrombotic function of AßPP primarily resides in the KPI activity of the protein.

The impact of human and mouse differences in NOS2 gene expression on the brain's redox and immune environment.

BACKGROUND: Mouse models are used in the study of human disease. Despite well-known homologies, the difference in immune response between mice and humans impacts the application of data derived from mice to human disease outcomes. Nitric oxide synthase-2 (NOS2) is a key gene that displays species-specific outcomes via altered regulation of the gene promoter and via post-transcriptional mechanisms in humans that are not found in mice. The resulting levels of NO produced by activation of human NOS2 are different from the levels of NO produced by mouse Nos2. Since both tissue redox environment and immune responsiveness are regulated by the level of NO and its interactions, we investigated the significance of mouse and human differences on brain oxidative stress and on immune activation in HuNOS2tg/mNos2-/- mice that express the entire human NOS2 gene and that lack a functional mNos2 compared to wild type (WT) mice that express normal mNos2. METHODS/RESULTS: Similarly to human, brain tissue from HuNOS2tg/mNos2-/- mice showed the presence of a NOS2 gene 3'UTR binding site. We also identified miRNA-939, the binding partner for this site, in mouse brain lysates and further demonstrated reduced levels of nitric oxide (NO) typical of the human immune response on injection with lipopolysaccharide (LPS). HuNOS2tg/mNos2-/- brain samples were probed for characteristic differences in redox and immune gene profiles compared to WT mice using gene arrays. Selected genes were also compared against mNos2-/- brain lysates. Reconstitution of the human NOS2 gene significantly altered genes that encode multiple anti-oxidant proteins, oxidases, DNA repair, mitochondrial proteins and redox regulated immune proteins. Expression levels of typical pro-inflammatory, anti-inflammatory and chemokine genes were not significantly different with the exception of increased TNFα and Ccr1 mRNA expression in the HuNOS2tg/mNos2-/- mice compared to WT or mNos2-/- mice. CONCLUSIONS: NO is a principle factor in establishing the tissue redox environment and changes in NO levels impact oxidative stress and immunity, both of which are primary characteristics of neurodegenerative diseases. The HuNOS2tg/mNos2-/- mice provide a potentially useful mechanism to address critical species- specific immune differences that can impact the study of human diseases.

Longitudinal study of differential protein expression in an Alzheimer's mouse model lacking inducible nitric oxide synthase.

Alzheimer's disease (AD) is a complex neurodegenerative process that involves altered brain immune, neuronal and metabolic functions. Understanding the underlying mechanisms has relied on mouse models that mimic components of AD pathology. We used gel-free, label-free LC-MS/MS to quantify protein and phosphopeptide levels in brains of APPSwDI/NOS2-/- (CVN-AD) mice. CVN-AD mice show a full spectrum of AD-like pathology, including amyloid deposition, hyperphosphorylated and aggregated tau, and neuronal loss that worsens with age. Tryptic digests, with or without phosphopeptide enrichment on an automated titanium dioxide LC system, were separated by online two-dimensional LC and analyzed on a Waters Synapt G2 HDMS, yielding relative expression for >950 proteins and >1100 phosphopeptides. Among differentially expressed proteins were known markers found in humans with AD, including GFAP and C1Q. Phosphorylation of connexin 43, not previously described in AD, was increased at 42 weeks, consistent with dysregulation of gap junctions and activation of astrocytes. Additional alterations in phosphoproteins suggests dysregulation of mitochondria, synaptic transmission, vesicle trafficking, and innate immune pathways. These data validate the CVN-AD mouse model of AD, identify novel disease and age-related changes in the brain during disease progression, and demonstrate the utility of integrating unbiased and phosphoproteomics for understanding disease processes in AD.

Fine mapping of the amyloid ß-protein binding site on myelin basic protein.

The assembly and deposition of amyloid ß-protein (Aß) in brain is a key pathological feature of Alzheimer's disease and related disorders. Factors have been identified that can either promote or inhibit Aß assembly in brain. We previously reported that myelin basic protein (MBP) is a potent inhibitor of Aß fibrillar assembly [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., et al. (2009) Biochemistry 48, 4720-4727]. Moreover, the region on MBP responsible for this activity was localized to the 64 N-terminal amino acids (MBP1-64) [Liao, M. C., et al. (2010) J. Biol. Chem. 285, 35590-35598]. In the study presented here, we sought to further define the site on MBP1-64 involved in this activity. Deletion mapping studies showed that the C-terminal region (residues 54-64) is required for the ability of MBP1-64 to bind Aß and inhibit fibril assembly. Alanine scanning mutagenesis revealed that amino acids K54, R55, G56, and K59 within MBP1-64 are important for both Aß binding and inhibition of fibril assembly as assessed by solid phase binding, thioflavin T binding and fluorescence, and transmission electron microscopy studies. Strong spectral shifts are observed by solution nuclear magnetic resonance spectroscopy of specific N-terminal residues (E3, R5, D7, E11, and Q15) of Aß42 upon the interaction with MBP1-64. Although the C-terminal region of MBP1-64 is required for interactions with Aß, a synthetic MBP50-64 peptide was itself devoid of activity. These studies identify key residues in MBP and Aß involved in their interactions and provide structural insight into how MBP regulates Aß fibrillar assembly.

Nitric oxide-mediated regulation of ß-amyloid clearance via alterations of MMP-9/TIMP-1.

Fibrillar amyloid plaques are largely composed of amyloid-beta (Aß) peptides that are metabolized into products, including Aß1-16, by proteases including matrix metalloproteinase 9 (MMP-9). The balance between production and degradation of Aß proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP-9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP-9/TIMP-1 balance. We show NO-mediated increased MMP-9/TIMP-1 ratios enhanced the degradation of fibrillar Aß in vitro, which was abolished when silenced for MMP-9 protein translation. The in vivo relationship between MMP-9, NO and Aß degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP-9 mediated changes, we generated an antibody recognizing the Aß1-16 fragment, and used mass spectrometry multi-reaction monitoring assay for detection of immunoprecipitated Aß1-16 peptides. Aß1-16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP-1 increased in the APPSwDI/NOS2(-/-) mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.

N-terminal domain of myelin basic protein inhibits amyloid beta-protein fibril assembly.

Accumulation of amyloid ß-protein (Aß) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aß peptides readily form ß-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aß 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 Aß and inhibited Aß 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 Aß fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aß is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aß fibrillogenesis. Under conditions comparable with those used for Aß, 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 Aß in primary cortical neurons. Our findings suggest that inhibition of Aß 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.

Myelin basic protein binds to and inhibits the fibrillar assembly of Abeta42 in vitro.

The deposition of amyloid beta-protein (Abeta) fibrils into plaques within the brain parenchyma and along cerebral blood vessels is a hallmark of Alzheimer's disease. Abeta peptides are produced through the successive cleavage of the Abeta precursor protein by beta- and gamma-secretase, producing peptides between 39 and 43 amino acids in length. The most common of these are Abeta40 (the most abundant) and Abeta42. Abeta42 is more fibrillogenic than Abeta40 and has been implicated in early Abeta plaque deposition. Our previous studies determined that myelin basic protein (MBP) was capable of inhibiting fibril formation of a highly fibrillogenic Abeta peptide containing both E22Q (Dutch) and D23N (Iowa) mutations associated with familial forms of cerebral amyloid angiopathy [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961]. In this study, we show through a combination of biochemical and ultrastructural techniques that MBP is also capable of inhibiting the beta-sheet fibrillar assembly of the normal Abeta42 peptide. These findings suggest that MBP may play a role in regulating the deposition of Abeta42 and thereby also may regulate the early formation of amyloid plaques in Alzheimer's disease.

Inhibition of familial cerebral amyloid angiopathy mutant amyloid beta-protein fibril assembly by myelin basic protein.

Deposition of fibrillar amyloid beta-protein (Abeta) in the brain is a prominent pathological feature of Alzheimer disease and related disorders, including familial forms of cerebral amyloid angiopathy (CAA). Mutant forms of Abeta, including Dutch- and Iowa-type Abeta, which are responsible for familial CAA, deposit primarily as fibrillar amyloid along the cerebral vasculature and are either absent or present only as diffuse non-fibrillar plaques in the brain parenchyma. Despite the lack of parenchymal fibril formation in vivo, these CAA mutant Abeta peptides exhibit a markedly increased rate and extent of fibril formation in vitro compared with wild-type Abeta. Based on these conflicting observations, we sought to determine whether brain parenchymal factors that selectively interact with and modulate CAA mutant Abeta fibril assembly exist. Using a combination of immunoaffinity chromatography and mass spectrometry, we identified myelin basic protein (MBP) as a prominent brain parenchymal factor that preferentially binds to CAA mutant Abeta compared with wild-type Abeta. Surface plasmon resonance measurements confirmed that MBP bound more tightly to Dutch/Iowa CAA double mutant Abeta than to wild-type Abeta. Using a combination of biochemical and ultrastructural techniques, we found that MBP inhibited the fibril assembly of CAA mutant Abeta. Together, these findings suggest a possible role for MBP in regulating parenchymal fibrillar Abeta deposition in familial CAA.