Mary Jo LaDu

Oligomeric and Fibrillar Species of Amyloid-β Peptides Differentially Affect Neuronal Viability

Genetic evidence predicts a causative role for amyloid-β (Aβ) in Alzheimers disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of Aβ are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of Aβ-(1–42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and ∼40-fold more than unaggregated peptide, with oligomeric Aβ-(1–42)-induced inhibition significant at 10 nm. Under Aβ-(1–42) oligomer- and fibril-forming conditions, Aβ-(1–40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of Aβ-(1–42). We applied the aggregation protocols developed for wild type Aβ-(1–42) to Aβ-(1–42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type Aβ-(1–42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type Aβ-(1–42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar Aβ-(1–42) are useful in distinguishing the structural and functional differences between Aβ-(1–42) and Aβ-(1–40) and genetic mutations of Aβ-(1–42).

In vitro characterization of conditions for amyloid-β peptide oligomerization and fibrillogenesis

Extensive research causally links amyloid-β peptide (Aβ) to Alzheimers disease, although the pathologically relevant Aβ conformation remains unclear. Aβ spontaneously aggregates into the fibrils that deposit in senile plaques. However, recent in vivo and in vitroreports describe a potent biological activity for oligomeric assemblies of Aβ. To consistently prepare in vitro oligomeric and fibrillar forms of Aβ1–42, a detailed knowledge of how solution parameters influence structure is required. This manuscript represents the first study using a single chemically and structurally homogeneous unaggregated starting material to demonstrate that the formation of oligomers, fibrils, and fibrillar aggregates is determined by time, concentration, temperature, pH, ionic strength, and Aβ species. We recently reported that oligomers inhibit neuronal viability 10-fold more than fibrils and ∼40-fold more than unaggregated peptide, with oligomeric Aβ1–42-induced neurotoxicity significant at 10 nm. In addition, we were able to differentiate by structure and neurotoxic activity wild-type Aβ1–42 from isoforms containing familial mutations (Dahlgren, K. N., Manelli, A. M., Stine, W. B., Jr., Baker, L. K., Krafft, G. A., and LaDu, M. J. (2002) J. Biol. Chem. 277, 32046–32053). Understanding the biological role of specific Aβ conformations may define the link between Aβ and Alzheimers disease, re-focusing therapeutic approaches by identifying the pernicious species of Aβ ultimately responsible for the cognitive dysfunction that defines the disease.

Nascent Astrocyte Particles Differ from Lipoproteins in CSF

Abstract: Little is known about lipid transport and metabolism in the brain. As a further step toward understanding the origin and function of CNS lipoproteins, we have characterized by size and density fractionation lipoprotein particles from human CSF and primary cultures of rat astrocytes. The fractions were analyzed for esterified and free cholesterol, triglyceride, phospholipid, albumin, and apolipoproteins (apo) E, AI, AII, and J. As determined by lipid and apolipoprotein profiles, gel electrophoresis, and electron microscopy, nascent astrocyte particles contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ. In contrast, CSF lipoproteins are the size and density of plasma high‐density lipoprotein, contain the core lipid, esterified cholesterol, and are spherical. CSF lipoproteins were heterogeneous in apolipoprotein content with apoE, the most abundant apolipoprotein, localized to the largest particles, apoAI and apoAII localized to progressively smaller particles, and apoJ distributed relatively evenly across particle size. There was substantial loss of protein from both CSF and astrocyte particles after density centrifugation compared with gel‐filtration chromatography. The differences between lipoproteins secreted by astrocytes and present in CSF suggest that in addition to delivery of their constituents to cells, lipoprotein particles secreted within the brain by astrocytes may have the potential to participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF. Study of the functional properties of both astrocyte‐secreted and CSF lipoproteins isolated by techniques that preserve native particle structure may also provide insight into the function of apoE in the pathophysiology of specific neurological diseases such as Alzheimers disease.

Glial Fibrillary Acidic Protein–Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins

The ε4 allele of apolipoprotein E (apoE) is associated with increased risk for Alzheimer’s disease (AD) and poor outcome after brain injury. In the CNS, apoE is expressed by glia, predominantly astrocytes. To define the potential biological functions of different human apoE isoforms produced within the brain, transgenic mice were generated in which human apoE3 and apoE4 expression is under control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. These animals were then bred back to apoE knock-out mice. Human apoE protein is found within astrocytes and the neuropil throughout development and into the adult period, as assessed by immunocytochemistry and immunoblot analysis in several GFAP-apoE3 and E4 lines. Cultured astrocytes from these mice secrete apoE3 and apoE4 in lipoproteins that are high-density lipoprotein-like in size. When primary hippocampal neurons are grown in the presence of astrocyte monolayers derived from these transgenic mice, there is significantly greater neurite outgrowth from neurons grown in the presence of apoE3-secreting astrocytes compared with apoE4-secreting or apoE knock-out astrocytes. These effects are not dependent on direct astrocyte–neuron contact and appear to require the low-density lipoprotein receptor-related protein. These data suggest that astrocyte-secreted, apoE3-containing lipoproteins have different biological effects than apoE4-containing lipoproteins. In addition to providing information regarding the role of astrocyte-secreted apoE lipoproteins in the normal brain, these animals will also be useful in models of both AD and CNS injury.

Lipoproteins in the central nervous system

Abstract: Although the synthesis and metabolism of plasma lipoproteins are well characterized, little is known about lipid delivery and clearance within the central nervous system (CNS). Our work has focused on characterizing the lipoprotein particles present in the cerebrospinal fluid (CSF) and the nascent particles secreted by astrocytes. In addition to carrying lipids, we have found that β‐amyloid (Ab) associates with lipoproteins, including the discoidal particles secreted by cultured astrocytes and the spherical lipoproteins found in CSF. We believe that association with lipoproteins provides a means of transport and clearance for Aβ. This process may be further influenced by an interaction between Ab and apoprotein E (apoE), the primary protein component of CNS lipoproteins. Specifically, we have investigated the formation and physiologic relevance of a SDS‐stable complex between apoE and Aβ. In biochemical assays, native apoE2 and E3 (associated with lipid particles) form an SDS‐stable complex with Ab that is 20‐fold more abundant than the apoE4:Aβ complex. In cell culture, native apoE3 but not E4 prevents Aβ‐induced neurotoxicity by a mechanism dependent on cell surface apoE receptors. In addition, apoE and the inhibition of apoE receptors prevent Aβ‐induced astrocyte activation. Therefore, we hypothesize that the protection from Aβ‐induced neurotoxicity afforded by apoE3 may result from clearance of the peptide by SDS‐stable apoE3:Aβ complex formation and uptake by apoE receptors.

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The ε4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer’s disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (−/−), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouseapoE (−/−) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles.ApoE (−/−) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (−/−) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.

ApoE4 Decreases Spine Density and Dendritic Complexity in Cortical Neurons In Vivo

The three human alleles of apolipoprotein E (APOE) differentially influence outcome after CNS injury and affect ones risk of developing Alzheimers disease (AD). It remains unclear how ApoE isoforms contribute to various AD-related pathological changes (e.g., amyloid plaques and synaptic and neuron loss). Here, we systematically examined whether apoE isoforms (E2, E3, E4) exhibit differential effects on dendritic spine density and morphology in APOE targeted replacement (TR) mice, which lack AD pathological changes. Using Golgi staining, we found age-dependent effects of APOE4 on spine density in the cortex. The APOE4 TR mice had significantly reduced spine density at three independent time points (4 weeks, 3 months, and 1 year, 27.7% ± 7.4%, 24.4% ± 8.6%, and 55.6% ± 10.5%, respectively) compared with APOE3 TR mice and APOE2 TR mice. Additionally, in APOE4 TR mice, shorter spines were evident compared with other APOE TR mice at 1 year. APOE2 TR mice exhibited longer spines as well as significantly increased apical dendritic arborization in the cortex compared with APOE4 and APOE3 TR mice at 4 weeks. However, there were no differences in spine density across APOE genotypes in hippocampus. These findings demonstrate that apoE isoforms differentially affect dendritic complexity and spine formation, suggesting a role for APOE genotypes not only in acute and chronic brain injuries including AD, but also in normal brain functions.

Cognitive effects of cell-derived and synthetically derived Aβ oligomers.

Soluble forms of amyloid-β peptide (Aβ) are a molecular focus in Alzheimers disease research. Soluble Aβ dimers (≈8 kDa), trimers (≈12 kDa), tetramers (≈16 kDa) and Aβ*56 (≈56 kDa) have shown biological activity. These Aβ molecules have been derived from diverse sources, including chemical synthesis, transfected cells, and mouse and human brain, leading to uncertainty about toxicity and potency. Herein, synthetic Aβ peptide-derived oligomers, cell- and brain-derived low-n oligomers, and Aβ*56, were injected intracerebroventricularly (icv) into rats assayed under the Alternating Lever Cyclic Ratio (ALCR) cognitive assay. Cognitive deficits were detected at 1.3 μM of synthetic Aβ oligomers and at low nanomolar concentrations of cell-secreted Aβ oligomers. Trimers, from transgenic mouse brain (Tg2576), did not cause cognitive impairment at any dose tested, whereas Aβ*56 induced concentration-dependent cognitive impairment at 0.9 and 1.3μM. Thus, while multiple forms of Aβ have cognition impairing activity, there are significant differences in effective concentration and potency.

A dual role for apolipoprotein e in neuroinflammation: anti- and pro-inflammatory activity.

Chronically activated glia associated with amyloid plaques might contribute to neuronal dysfunction in Alzheimer’s disease (AD) through generation of neuroinflammatory molecules. Apolipoprotein E (apoE), also found associated with amyloid plaques, has been hypothesized to serve an anti-inflammatory role in the CNS through its ability to modulate β-amyloid (Aβ)-induced glial activation. To further characterize the effect of apoE on inflammation, we examined the ability of exogenously added human apoE3 and apoE4 to modulate neuroinflammatory responses of cultured rat glia. Apolipoprotein E3 (apoE3) and apoE4 suppressed oligomeric Aβ-induced production of inducible nitric oxide synthase and cyclo-oxygenase-2, supporting an anti-inflammatory role for apoE. Exogenous apoE also inhibited Aβ-induced production of endogenous apoE. However, exogenous apoE in the absence of Aβ stimulated production of the pro-inflammatory cytokine interleukin-1β in an isoform-dependent manner, with apoE4 inducing a significantly greater response than apoE3. These data support the idea that Aβ stimulation of glial apoE limits neuroinflammation but that overproduction of apoE by activated glia might exacerbate inflammation. In addition, the observation that apoE4 has more robust pro-inflammatory activity than apoE3 provides a mechanistic link between the APOE4 allele and AD, and suggests potential apoE-based therapeutic strategies.

Overexpression of Human Apolipoprotein A-I Preserves Cognitive Function and Attenuates Neuroinflammation and Cerebral Amyloid Angiopathy in a Mouse Model of Alzheimer Disease

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-β (Aβ) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-β precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aβ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aβ-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.