Judianne Davis

LRP/Amyloid β-Peptide Interaction Mediates Differential Brain Efflux of Aβ Isoforms

Abstract LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimers disease (AD). Here, we report amyloid β-peptide Aβ40 binds to immobilized LRP clusters II and IV with high affinity (K d = 0.6–1.2 nM) compared to Aβ42 and mutant Aβ, and LRP-mediated Aβ brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high β sheet content in Aβ and deletion of the receptor-associated protein gene. Despite low Aβ production in the brain, transgenic mice expressing low LRP-clearance mutant Aβ develop robust Aβ cerebral accumulations much earlier than Tg-2576 Aβ-overproducing mice. While Aβ does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations >1 μM, consistent with reduced brain capillary LRP levels in Aβ-accumulating transgenic mice, AD, and patients with cerebrovascular β-amyloidosis. Thus, low-affinity LRP/Aβ interaction and/or Aβ-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Aβ.

Structural conversion of neurotoxic amyloid-[beta]1-42 oligomers to fibrils

The amyloid-β1–42 (Aβ42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimers disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the β-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Aβ42 oligomers to fibrils involves the association of these loosely aggregated strands into β-sheets whose individual β-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.

Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid.

Cerebral microvascular amyloid β protein (Aβ) deposition and associated neuroinflammation is increasingly recognized as an important component leading to cognitive impairment in Alzheimers disease and related cerebral amyloid angiopathy disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Aβ precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits and exhibit robust neuroinflammation. In the present study, we investigated the effect of the anti-inflammatory drug minocycline on Aβ accumulation, neuroinflammation, and behavioral deficits in Tg-SwDI mice. Twelve-month-old mice were treated with saline or minocycline by intraperitoneal injection every other day for a total of 4 weeks. During the final week of treatment, the mice were tested for impaired learning and memory. Brains were then harvested for biochemical and immunohistochemical analysis. Minocycline treatment did not alter the cerebral deposition of Aβ or the restriction of fibrillar amyloid to the cerebral microvasculature. Similarly, minocycline-treated Tg-SwDI mice exhibited no change in the levels of total Aβ, the ratios of Aβ40 and Aβ42, or the amounts of soluble, insoluble, or oligomeric Aβ compared with the saline-treated control Tg-SwDI mice. In contrast, the numbers of activated microglia and levels of interleukin-6 were significantly reduced in minocycline-treated Tg-SwDI mice compared with saline-treated Tg-SwDI mice. In addition, there was a significant improvement in behavioral performance of the minocycline-treated Tg-SwDI mice. These finding suggest that anti-inflammatory treatment targeted for cerebral microvascular amyloid-induced microglial activation can improve cognitive deficits without altering the accumulation and distribution of Aβ.

Cerebral Microvascular Amyloid β Protein Deposition Induces Vascular Degeneration and Neuroinflammation in Transgenic Mice Expressing Human Vasculotropic Mutant Amyloid β Precursor Protein

Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimers disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid beta-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Abeta exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Abeta accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Abeta deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1beta and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.

Progression of Amyloid Pathology to Alzheimer's Disease Pathology in an Amyloid Precursor Protein Transgenic Mouse Model by Removal of Nitric Oxide Synthase 2

Alzheimers disease (AD) is characterized by three primary pathologies in the brain: amyloid plaques, neurofibrillary tangles, and neuron loss. Mouse models have been useful for studying components of AD but are limited in their ability to fully recapitulate all pathologies. We crossed the APPSwDI transgenic mouse, which develops amyloid β (Aβ)-protein deposits only, with a nitric oxide synthase 2 (NOS2) knock-out mouse, which develops no AD-like pathology. APPSwDI/NOS2−/− mice displayed impaired spatial memory compared with the APPSwDI mice, yet they have unaltered levels of Aβ. APPSwDI mice do not show tau pathology, whereas APPSwDI/NOS2−/− mice displayed extensive tau pathology associated with regions of dense microvascular amyloid deposition. Also, APPSwDI mice do not have any neuron loss, whereas the APPSwDI/NOS2−/− mice have significant neuron loss in the hippocampus and subiculum. Neuropeptide Y neurons have been shown to be particularly vulnerable in AD. These neurons appear to be particularly vulnerable in the APPSwDI/NOS2−/− mice as we observe a dramatic reduction in the number of NPY neurons in the hippocampus and subiculum. These data show that removal of NOS2 from an APP transgenic mouse results in development of a much greater spectrum of AD-like pathology and behavioral impairments.

Conformational Differences between Two Amyloid β Oligomers of Similar Size and Dissimilar Toxicity

Background: The Alzheimer Aβ peptide assembles into multiple small oligomers that are cytotoxic. Results: Increased solvent exposure of hydrophobic residues within non-fibrillar Aβ oligomers of similar size increases cytotoxicity. Conclusion: Aβ non-fibrillar oligomers display size-independent differences in toxicity that are strongly influenced by oligomer conformation. Significance: Identifying the conformational determinants of Aβ-mediated toxicity is critical to understand and treat Alzheimer disease. 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.

Amyloid Reduction by Amyloid-β Vaccination Also Reduces Mouse Tau Pathology and Protects from Neuron Loss in Two Mouse Models of Alzheimer's Disease

Shown to lower amyloid deposits and improve cognition in APP transgenic mouse models, immunotherapy appears to be a promising approach for the treatment of Alzheimers disease (AD). Due to limitations in available animal models, however, it has been unclear whether targeting amyloid is sufficient to reduce the other pathological hallmarks of AD—namely, accumulation of pathological, nonmutated tau and neuronal loss. We have now developed two transgenic mouse models (APPSw/NOS2−/− and APPSwDI/NOS2−/−) that more closely model AD. These mice show amyloid pathology, hyperphosphorylated and aggregated normal mouse tau, significant neuron loss, and cognitive deficits. Aβ1–42 or KLH vaccinations were started in these animals at 12 months, when disease progression and cognitive decline are well underway, and continued for 4 months. Vaccinated APPSwDI/NOS2−/− mice, which have predominantly vascular amyloid pathology, showed a 30% decrease in brain Aβ and a 35–45% reduction in hyperphosphorylated tau. Neuron loss and cognitive deficits were partially reduced. In APPSw/NOS2−/− vaccinated mice, brain Aβ was reduced by 65–85% and hyperphosphorylated tau by 50–60%. Furthermore, neurons were completely protected, and memory deficits were fully reversed. Microhemorrhage was observed in all vaccinated APPSw/NOS2−/− mice and remains a significant adverse event associated with immunotherapy. Nevertheless, by providing evidence that reducing amyloid pathology also reduces nonmutant tau pathology and blocks neuron loss, these data support the development of amyloid-lowering therapies for disease-modifying treatment of AD.

The Effects of NOS2 Gene Deletion on Mice Expressing Mutated Human AβPP

Nitric oxide synthase 2 (NOS2) and its gene product, inducible NOS (iNOS) play an important role in neuroinflammation by generating nitric oxide (NO), a critical signaling and redox factor in the brain. Although NO is associated with tissue damage, it can also promote cell survival. We hypothesize that during long-term exposure to amyloid-beta (Abeta) in Alzheimers disease (AD), NO levels fall in the brain to a threshold at which the protective effects of NO cannot be sustained, promoting Abeta mediated damage. Two new mouse models of AD have been developed that utilize this concept of NOs action. These mice express human amyloid-beta protein precursor (AbetaPP) mutations that generate Abeta peptides on a mouse NOS2 knockout background. The APP/NOS2(-/-) bigenic mice progress from Abeta production and amyloid deposition to hyperphosphorylated normal mouse tau at AD-associated epitopes, aggregation and redistribution of tau to somatodendritic regions of neurons and significant neuronal loss including loss of interneurons. This AD-like pathology is accompanied by robust behavioral changes. As APP/NOS2(-/-) bigenic mice more fully model the human AD disease pathology, they may serve as a tool to better understand disease progression in AD and the role of NO in altering chronic neurological disease processes.

Reducing Cerebral Microvascular Amyloid-β Protein Deposition Diminishes Regional Neuroinflammation in Vasculotropic Mutant Amyloid Precursor Protein Transgenic Mice

Cerebral microvascular amyloid-β (Aβ) protein deposition is emerging as an important contributory factor to neuroinflammation and dementia in Alzheimers disease and related familial cerebral amyloid angiopathy disorders. In particular, cerebral microvascular amyloid deposition, but not parenchymal amyloid, is more often correlated with dementia. Recently, we generated transgenic mice (Tg-SwDI) expressing the vasculotropic Dutch (E693Q)/Iowa (D694N) mutant human Aβ precursor protein in brain that accumulate abundant cerebral microvascular fibrillar amyloid deposits. In the present study, our aim was to assess how the presence or absence of fibrillar Aβ deposition in the cerebral microvasculature affects neuroinflammation in Tg-SwDI mice. Using Tg-SwDI mice bred onto an apolipoprotein E gene knock-out background, we found a strong reduction of fibrillar cerebral microvascular Aβ deposition, which was accompanied by a sharp decrease in microvascular-associated neuroinflammatory cells and interleukin-1β levels. Quantitative immunochemical measurements showed that this reduction of the neuroinflammation occurred in the absence of lowering the levels of total Aβ40/Aβ42 or soluble Aβ oligomers in brain. These findings suggest that specifically reducing cerebral microvascular fibrillar Aβ deposition, in the absence of lowering either the total amount of Aβ or soluble Aβ oligomers in brain, may be sufficient to ameliorate microvascular amyloid-associated neuroinflammation.

Regulation of ischemic cell death by glucocorticoids and adrenocorticotropic hormone

Transient global ischemia results in delayed selective neuronal death of hippocampal CA1 pyramidal cells. Glucocorticoids increase and adrenalectomy decreases the rate of neuronal death; however, they also produce changes in brain temperature, serum glucose and adrenocorticotropic hormone levels. In order to understand the role of glucocorticoids in regulating ischemic cell death, we studied RU 38486, a glucocorticoid receptor blocker, and Org 2766, a non-steroidogenic adrenocorticotropic hormone 4-9 analog. Male Mongolian gerbils were subjected to 5 min of bilateral carotid artery occlusion under a controlled temperature environment (37.0-38.0 degrees C). Animals were injected with either physiological saline, Org 2766 (10 microg/kg/24 h) or RU 38486 (50 mg/kg/8 h), beginning just prior to the occlusion until killing at either day 4 or 7. Blood was collected for serum glucose and cortisol analysis. Damage was evaluated by blinded counts of CAI neurons. Both RU 38486 and Org 2766 treatment significantly (P<0.004) reduced hippocampal CA1 damage at day 4, but not on day 7. While RU 38486 raised serum cortisol and adrenocorticotropic hormone levels, neither treatment affected temperature or serum glucose. The fact that RU 38486 mimicked adrenalectomy without changing temperature suggests that the decreased rate of cell death resulted from either removal of glucocorticoids or increases in adrenocorticotropic hormone. The ability of Org 2766 to affect this rate strongly suggests that adrenocorticotropic hormone is the active regulatory hormone rather than glucocorticoids. While both RU 38486 and Org 2766 prolong the survival of CA1 neurons after transient global ischemia, only RU 38486, which is available and tested in both animals and humans, can block the detrimental effects of post-ischemia glucocorticoid elevations. Thus, the administration of RU 38486 may be a practical adjunct to other neuroprotective agents for victims of cardiac arrest, anesthetic accidents or drowning.