John D. Fryer

Human Apolipoprotein E4 Alters the Amyloid-β 40:42 Ratio and Promotes the Formation of Cerebral Amyloid Angiopathy in an Amyloid Precursor Protein Transgenic Model

Alzheimers disease (AD) is characterized by the aggregation and deposition of the normally soluble amyloid-β (Aβ) peptide in the extracellular spaces of the brain as parenchymal plaques and in the walls of cerebral vessels as cerebral amyloid angiopathy (CAA). CAA is a common cause of brain hemorrhage and is found in most patients with AD. As in AD, the ϵ4 allele of the apolipoprotein E (apoE) gene (APOE) is a risk factor for CAA. To determine the effect of human apoE on CAA in vivo, we bred human APOE3 and APOE4 “knock-in” mice to a transgenic mouse model (Tg2576) that develops amyloid plaques as well as CAA. The expression of both human apoE isoforms resulted in a delay in Aβ deposition of several months relative to murine apoE. Tg2576 mice expressing the more fibrillogenic murine apoE develop parenchymal amyloid plaques and CAA by 9 months of age. At 15 months of age, the expression of human apoE4 led to substantial CAA with very few parenchymal plaques, whereas the expression of human apoE3 resulted in almost no CAA or parenchymal plaques. Additionally, young apoE4-expressing mice had an elevated ratio of Aβ 40:42 in brain extracellular pools and a lower 40:42 ratio in CSF, suggesting that apoE4 results in altered clearance and transport of Aβ species within different brain compartments. These findings demonstrate that, once Aβ fibrillogenesis occurs, apoE4 favors the formation of CAA over parenchymal plaques and suggest that molecules or treatments that increase the ratio of Aβ 40:42 may favor the formation of CAA versus parenchymal plaques.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation–induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Purification and characterization of astrocyte-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice

The varepsilon4 allele of apolipoprotein E (apoE) is a genetic risk factor for Alzheimers disease (AD). In order to gain a better understanding of the molecular mechanisms by which apoE and possibly other apolipoproteins produced in the central nervous system (CNS) influence AD pathogenesis, we have purified and characterized the two most abundant apolipoproteins produced in the CNS, apoE and apoJ. We purified apoE and apoJ from primary cultures of mouse astrocytes, which were derived from transgenic mice expressing human apoE isoforms in the absence of mouse apoE. Utilizing antibody affinity columns, we were able to purify both human apoE3 and apoE4, as well as mouse apoJ-containing lipoproteins. Astrocyte-secreted human apoE was present in high density-like lipoproteins of three predominant sizes ranging from 8 to 15 nm in diameter. Mouse apoJ was in particles between 10 and 17 nm in diameter with a peak size range of approximately 11 nm. ApoE and apoJ were in distinct lipoproteins. Utilization of quick-freeze, deep-etch electron microscopy revealed the apoE particles were discs while the apoJ particles were smaller and more irregular in appearance. The lipid composition of apoE particles was very different from those containing apoJ. ApoE-particles contained a similar mass of apoE and lipid, with cholesterol and phospholipid being about equal in mass per particle. ApoJ-particles were relatively lipid poor (three parts protein, one part lipid), with phospholipids being much more abundant than cholesterol. Detailed characterization of phospholipid composition by electrospray ionization mass spectrometry analysis revealed ethanolamine glycerophospholipids to be the most abundant phospholipid present in both apoE and apoJ particles. Analysis of cerebrospinal fluid from apoE3 and apoE4 transgenic mice revealed that human and mouse apoE were in particles the same size as those secreted by astrocytes. Further use of physiological preparations of CNS-derived lipoproteins may allow for a detailed understanding of the role of these molecules in the normal brain and in diseases such as AD.

A Synthetic Peptide Blocking the Apolipoprotein E/β-Amyloid Binding Mitigates β-Amyloid Toxicity and Fibril Formation in Vitro and Reduces β-Amyloid Plaques in Transgenic Mice

Alzheimer’s disease (AD) is associated with accumulation of β-amyloid (Aβ). A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele. ApoE can act as a pathological chaperone of Aβ, promoting its conformational transformation from soluble Aβ into toxic aggregates. We determined if blocking the apoE/Aβ interaction reduces Aβ load in transgenic (Tg) AD mice. The binding site of apoE on Aβ corresponds to residues 12 to 28. To block binding, we synthesized a peptide containing these residues, but substituted valine at position 18 to proline (Aβ12–28P). This changed the peptide’s properties, making it non-fibrillogenic and non-toxic. Aβ12–28P competitively blocks binding of full-length Aβ to apoE (IC50 = 36.7 nmol). Furthermore, Aβ12–28P reduces Aβ fibrillogenesis in the presence of apoE, and Aβ/apoE toxicity in cell culture. Aβ12–28P is blood-brain barrier-permeable and in AD Tg mice inhibits Aβ deposition. Tg mice treated with Aβ12–28P for 1 month had a 63.3% reduction in Aβ load in the cortex (P = 0.0043) and a 59.5% (P = 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice. Antibodies against Aβ were not detected in sera of treated mice; therefore the observed therapeutic effect of Aβ12–28P cannot be attributed to an antibody clearance response. Our experiments demonstrate that compounds blocking the interaction between Aβ and its pathological chaperones may be beneficial for treatment of β-amyloid deposition in AD.

Differences in the Aβ40/Aβ42 ratio associated with cerebrospinal fluid lipoproteins as a function of apolipoprotein E genotype

The ε4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimers disease (AD). ApoE, which is important for lipid metabolism, is also a major constituent of cerebrospinal fluid (CSF) lipoproteins (LPs). Although ApoE in the CSF is derived from the central nervous system, the relation between LP metabolism in plasma and CSF is not clear. Soluble amyloid‐β (Aβ) protein may normally be associated with CSF LPs. It is converted in AD to a fibrillar form in brain parenchyma. ApoE and CSF LPs may regulate this process. The purpose of this study was to characterize CSF LPs from healthy, cognitively normal, fasted, elderly individuals at different risk for AD based on ApoE genotype. Lipid composition of CSF LPs did not differ with ApoE genotype. Interestingly, plasma and CSF high‐density lipoprotein (HDL) cholesterol and apolipoprotein AI (ApoAI) levels were correlated. Importantly, as assessed by size‐exclusion chromatography, Aβ in CSF coeluted in fractions containing LPs and was influenced by ApoE genotype: E4‐positive subjects displayed significant elevations in Aβ40/Aβ42 ratios. These results suggest that plasma ApoAI/HDL levels can influence CSF ApoAI/HDL levels and that interactions between Aβ and central nervous system LPs may reflect changes in brain Aβ metabolism before the onset of clinical disease. Ann Neurol 2000;48:201–210

Matrix metalloproteinase-9 and spontaneous hemorrhage in an animal model of cerebral amyloid angiopathy.

We examined the potential role of the extra‐cellular matrix‐degrading enzyme, matrix metalloproteinase‐9 (MMP‐9), in the pathogenesis of cerebral amyloid angiopathy (CAA)‐induced spontaneous hemorrhage. The amyloid‐beta peptide (Aβ) induced the synthesis, release and activation of MMP‐9 in murine cerebral endothelial cells, resulting in increased extracellular matrix degradation. Furthermore, extensive MMP‐9 immunoreactivity was observed in CAA‐vessels with evidence of microhemorrhage in aged APPsw transgenic mice, but not detected in aged wild type or young APPsw mice. These results suggest that increased vascular MMP‐9 expression, stimulated by Aβ, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage in patients with CAA. Ann Neurol 2003;54:000–000

Matrix metalloproteinase-9 in cerebral-amyloid-angiopathy-related hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is one of the most recognized complications of cerebral amyloid angiopathy (CAA), but little is known about the molecular pathogenesis of this life-threatening complication. In this review, we present preliminary evidence which suggests that the extracellular-matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), may play a role in the development of spontaneous ICH resulting from CAA. The amyloid-beta peptide (Abeta) induced the synthesis, cellular release, and activation of MMP-9 in murine cerebral endothelial cells (CECs), resulting in increased extracellular matrix (ECM) degradation. Furthermore, in a mouse model of CAA (APPsw transgenic mice), MMP-9 immunoreactivity was observed in amyloid-laden cerebral vessels in aged APPsw mice but not in young APPsw or aged wild-type mice. More extensive MMP-9 immunostaining was present in amyloid-laden vessels with evidence of microhemorrhage. These results suggest that increased vascular MMP-9 expression, stimulated by Abeta, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage (ICH) in patients with CAA.

ApoAI Deficiency Results in Marked Reductions in Plasma Cholesterol But No Alterations in Amyloid-β Pathology in a Mouse Model of Alzheimer's Disease-Like Cerebral Amyloidosis

Epidemiological studies suggest links between cholesterol metabolism and Alzheimers disease (AD), with hypercholesterolemia associated with increased AD risk, and use of cholesterol-lowering drugs associated with decreased risk. Animal models using cholesterol-modifying dietary or pharmacological interventions demonstrate similar findings. Proposed mechanisms include effects of cholesterol on the metabolism of amyloid-beta (Abeta), the protein that deposits in AD brain. To investigate the effect of genetic alterations in plasma cholesterol on Abeta pathology, we crossed the PDAPP transgenic mouse model of AD-like cerebral amyloidosis to apolipoprotein AI-null mice that have markedly reduced plasma cholesterol levels due to a virtual absence of high density lipoproteins, the primary lipoprotein in mice. Interestingly and in contrast to models using non-physiological high fat diets or cholesterol-lowering drugs to modify plasma cholesterol, we observed no differences in Abeta pathology in PDAPP mice of the various apoAI genotypes despite robust differences in plasma cholesterol levels between the groups. Absence of apoAI also resulted in reductions in brain but not cerebrospinal fluid cholesterol, but had no effect on brain apolipoprotein E levels. These and other data suggest that it is perhaps the level of brain apolipoprotein E, not cholesterol per se, that plays a primary role in brain Abeta metabolism.

The Bad Seed in Alzheimer’s Disease

In this issue of Neuron, McGowan et al. report on a new mouse model of amyloid deposition as occurs in Alzheimers disease. Unlike previous models in which overexpression of the amyloid precursor protein results in amyloid plaque formation, McGowan et al. have produced mice that overexpress only Abeta40 or Abeta42 and prove that Abeta42 is critical for the formation of amyloid deposits in vivo.