Sophie Stukas

ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice

The cholesterol transpoter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Aβ metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce Aβ levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of Aβ to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.

High-Density Lipoproteins and Cerebrovascular Integrity in Alzheimer’s Disease

Cerebrovascular dysfunction significantly contributes to the clinical presentation and pathoetiology of Alzheimers disease (AD). Deposition and aggregation of β-amyloid (Aβ) within vascular smooth muscle cells leads to inflammation, oxidative stress, impaired vasorelaxation, and disruption of blood-brain barrier integrity. Midlife vascular risk factors, such as hypertension, cardiovascular disease, diabetes, and dyslipidemia, increase the relative risk for AD. These comorbidities are all characterized by low and/or dysfunctional high-density lipoproteins (HDL), which itself is a risk factor for AD. HDL performs a wide variety of critical functions in the periphery and CNS. In addition to lipid transport, HDL regulates vascular health via mediating vasorelaxation, inflammation, and oxidative stress and promotes endothelial cell survival and integrity. Here, we summarize clinical and preclinical data examining the involvement of HDL, originating from the circulation and from within the CNS, on AD and hypothesize potential synergistic actions between the two lipoprotein pools.

The Liver X Receptor Agonist GW3965 Improves Recovery from Mild Repetitive Traumatic Brain Injury in Mice Partly through Apolipoprotein E

Traumatic brain injury (TBI) increases Alzheimer’s disease (AD) risk and leads to the deposition of neurofibrillary tangles and amyloid deposits similar to those found in AD. Agonists of Liver X receptors (LXRs), which regulate the expression of many genes involved in lipid homeostasis and inflammation, improve cognition and reduce neuropathology in AD mice. One pathway by which LXR agonists exert their beneficial effects is through ATP-binding cassette transporter A1 (ABCA1)-mediated lipid transport onto apolipoprotein E (apoE). To test the therapeutic utility of this pathway for TBI, we subjected male wild-type (WT) and apoE−/− mice to mild repetitive traumatic brain injury (mrTBI) followed by treatment with vehicle or the LXR agonist GW3965 at 15 mg/kg/day. GW3965 treatment restored impaired novel object recognition memory in WT but not apoE−/− mice. GW3965 did not significantly enhance the spontaneous recovery of motor deficits observed in all groups. Total soluble Aβ40 and Aβ42 levels were significantly elevated in WT and apoE−/− mice after injury, a response that was suppressed by GW3965 in both genotypes. WT mice showed mild but significant axonal damage at 2 d post-mrTBI, which was suppressed by GW3965. In contrast, apoE−/− mice showed severe axonal damage from 2 to 14 d after mrTBI that was unresponsive to GW3965. Because our mrTBI model does not produce significant inflammation, the beneficial effects of GW3965 we observed are unlikely to be related to reduced inflammation. Rather, our results suggest that both apoE-dependent and apoE-independent pathways contribute to the ability of GW3965 to promote recovery from mrTBI.

Intravenously Injected Human Apolipoprotein A‐I Rapidly Enters the Central Nervous System via the Choroid Plexus

Background Brain lipoprotein metabolism is dependent on lipoprotein particles that resemble plasma high‐density lipoproteins but that contain apolipoprotein (apo) E rather than apoA‐I as their primary protein component. Astrocytes and microglia secrete apoE but not apoA‐I; however, apoA‐I is detectable in both cerebrospinal fluid and brain tissue lysates. The route by which plasma apoA‐I enters the central nervous system is unknown. Methods and Results Steady‐state levels of murine apoA‐I in cerebrospinal fluid and interstitial fluid are 0.664 and 0.120 μg/mL, respectively, whereas brain tissue apoA‐I is ≈10% to 15% of its levels in liver. Recombinant, fluorescently tagged human apoA‐I injected intravenously into mice localizes to the choroid plexus within 30 minutes and accumulates in a saturable, dose‐dependent manner in the brain. Recombinant, fluorescently tagged human apoA‐I accumulates in the brain for 2 hours, after which it is eliminated with a half‐life of 10.3 hours. In vitro, human apoA‐I is specifically bound, internalized, and transported across confluent monolayers of primary human choroid plexus epithelial cells and brain microvascular endothelial cells. Conclusions Following intravenous injection, recombinant human apoA‐I rapidly localizes predominantly to the choroid plexus. Because apoA‐I mRNA is undetectable in murine brain, our results suggest that plasma apoA‐I, which is secreted from the liver and intestine, gains access to the central nervous system primarily by crossing the blood–cerebrospinal fluid barrier via specific cellular mediated transport, although transport across the blood–brain barrier may also contribute to a lesser extent.

ABCA1 influences neuroinflammation and neuronal death

ATP-binding cassette transporter A1 (ABCA1) mediates cellular cholesterol efflux in the brain and influences whole brain cholesterol homeostasis. Activation of liver X receptors (LXRs), transcription factors that increase the expression of cholesterol transport genes including ABCA1, reduces neuroinflammation and pathology in neurodegenerative animal models suggesting that in addition to its involvement in cholesterol transport, ABCA1 may play a role in modulating the inflammatory response in the brain. We investigated the cell-type specific role of ABCA1 in neuroinflammation in vivo using mice specifically lacking brain ABCA1 (ABCA1(-B/-B)) as well as mice lacking neuronal (ABCA1(-N/-N)) and astrocytic (ABCA1(-Ast/-Ast)) ABCA1. ABCA1(-B/-B) mice exhibit cortical astrogliosis, increased inflammatory gene expression as well as activation of mitogen-activated protein kinases (MAPKs) following acute lipopolysaccharide (LPS) administration. Microglia cultured from ABCA1(-B/-B) mice exhibit augmented LPS-induced secretion of tumor necrosis factor α (TNFα) and decreased phagocytic activity, indicating an increase in a pro-inflammatory response. ABCA1(-N/-N) mice develop astrogliosis but show no change in inflammatory gene expression. Intriguingly, ABCA1(-Ast/-Ast) mice show neither astrogliosis nor elevated expression of inflammatory markers. Cortical apolipoprotein E (apoE) levels are reduced in ABCA1(-Ast/-Ast) but not in ABCA1(-N/-N) mice, providing in vivo evidence for the specific role of astrocyte ABCA1 in regulating brain apoE levels. Interestingly, cortical neuronal death is increased in 17month-old ABCA1(-B/-B) mice but not in ABCA1(-N/-N) or ABCA1(-Ast/-Ast) mice. Our findings suggest that coordinated ABCA1 activity across neurons and glial cells influences neuroinflammation and neurodegeneration.

Reconstituted high-density lipoproteins acutely reduce soluble brain Aβ levels in symptomatic APP/PS1 mice.

Many lines of evidence suggest a protective role for high-density lipoprotein (HDL) and its major apolipoprotein (apo)A-I in Alzheimers Disease (AD). HDL/apoA-I particles are produced by the liver and intestine and, in addition to removing excess cholesterol from the body, are increasingly recognized to have vasoprotective functions. Here we tested the ability of reconstituted HDL (rHDL) consisting of human apoA-I reconstituted with soy phosphatidylcholine for its ability to lower amyloid beta (Aβ) levels in symptomatic APP/PS1 mice, a well-characterized preclinical model of amyloidosis. Animals were treated intravenously either with four weekly doses (chronic study) or a single dose of 60mg/kg of rHDL (acute study). The major finding of our acute study is that soluble brain Aβ40 and Aβ42 levels were significantly reduced within 24h of a single dose of rHDL. By contrast, no changes were observed in our chronic study with respect to soluble or deposited Aβ levels in animals assessed 7days after the final weekly dose of rHDL, suggesting that beneficial effects diminish as rHDL is cleared from the body. Further, rHDL-treated animals showed no change in amyloid burden, cerebrospinal fluid (CSF) Aβ levels, neuroinflammation, or endothelial activation in the chronic study, suggesting that the pathology-modifying effects of rHDL may indeed be acute and may be specific to the soluble Aβ pool. That systemic administration of rHDL can acutely modify brain Aβ levels provides support for further investigation of the therapeutic potential of apoA-I-based agents for AD. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1.

Lipoprotein metabolism in the central nervous system (CNS) is based on high-density lipoprotein-like particles that use apoE as their predominant apolipoprotein rather than apoA-I. Although apoA-I is not expressed in astrocytes and microglia, which produce CNS apoE, apoA-I is reported to be expressed in porcine brain capillary endothelial cells and also crosses the blood-brain barrier (BBB). These mechanisms allow apoA-I to reach concentrations in cerebrospinal fluid (CSF) that are approximately 0.5% of its plasma levels. Recently, apoA-I has been shown to enhance cognitive function and reduce cerebrovascular amyloid deposition in Alzheimers Disease (AD) mice, raising questions about the regulation and function of apoA-I in the CNS. Peripheral apoA-I metabolism is highly influenced by ABCA1, but less is known about how ABCA1 regulates CNS apoA-I. We report that ABCA1 deficiency leads to greater retention of apoA-I in the CNS than in the periphery. Additionally, treatment of symptomatic AD mice with GW3965, an LXR agonist that stimulates ABCA1 expression, increases apoA-I more dramatically in the CNS compared to the periphery. Furthermore, GW3965-mediated up-regulation of CNS apoA-I is independent of ABCA1. Our results suggest that apoA-I may be regulated by distinct mechanisms on either side of the BBB and that apoA-I may serve to integrate peripheral and CNS lipid metabolism. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

An ABCA1-independent pathway for recycling a poorly lipidated 8.1 nm apolipoprotein E particle from glia

Lipid transport in the brain is coordinated by glial-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. Here we show that apoE is secreted from wild-type (WT) primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Negative-staining electron microscropy (EM) revealed rouleaux, suggesting a discoidal structure. Potassium bromide (KBr) density gradient ultracentrifugation showed that all subspecies, except an 8.1 nm particle, were lipidated. Glia lacking the cholesterol transporter ABCA1 secreted only 8.1 nm particles, which were poorly lipidated and nondiscoidal but could accept lipids to form the full repertoire of WT apoE particles. Receptor-associated-protein (RAP)-mediated inhibition of apoE receptor function blocked appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor (LDLR) reduced the level of 8.1 nm particle production by approximately 90%, suggesting that apoE is preferentially recycled through the LDLR. Finally, apoA-I stimulated secretion of 8.1 nm particles in a dose-dependent manner. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways and that a greater understanding of these mechanisms may be relevant to several neurological disorders.

Clearance of beta-amyloid is facilitated by apolipoprotein E and circulating high-density lipoproteins in bioengineered human vessels

Amyloid plaques, consisting of deposited beta-amyloid (Aβ), are a neuropathological hallmark of Alzheimer’s Disease (AD). Cerebral vessels play a major role in AD, as Aβ is cleared from the brain by pathways involving the cerebrovasculature, most AD patients have cerebrovascular amyloid (cerebral amyloid angiopathy (CAA), and cardiovascular risk factors increase dementia risk. Here we present a notable advance in vascular tissue engineering by generating the first functional 3-dimensioinal model of CAA in bioengineered human vessels. We show that lipoproteins including brain (apoE) and circulating (high-density lipoprotein, HDL) synergize to facilitate Aβ transport across bioengineered human cerebral vessels. These lipoproteins facilitate Aβ42 transport more efficiently than Aβ40, consistent with Aβ40 being the primary species that accumulates in CAA. Moreover, apoE4 is less effective than apoE2 in promoting Aβ transport, also consistent with the well-established role of apoE4 in Aβ deposition in AD.

Defining the biomechanical and biological threshold of murine mild traumatic brain injury using CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration)

ABSTRACT CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) is a recently described animal model of traumatic brain injury (TBI) that primarily produces diffuse axonal injury (DAI) characterized by white matter inflammation and axonal damage. CHIMERA was specifically designed to reliably generate a variety of TBI severities using precise and quantifiable biomechanical inputs in a nonsurgical user‐friendly platform. The objective of this study was to define the lower limit of single impact mild TBI (mTBI) using CHIMERA by characterizing the dose‐response relationship between biomechanical input and neurological, behavioral, neuropathological and biochemical outcomes. Wild‐type male mice were subjected to a single CHIMERA TBI using six impact energies ranging from 0.1 to 0.7 J, and post‐TBI outcomes were assessed over an acute period of 14 days. Here we report that single TBI using CHIMERA induces injury dose‐ and time‐dependent changes in behavioral and neurological deficits, axonal damage, white matter tract microgliosis and astrogliosis. Impact energies of 0.4 J or below produced no significant phenotype (subthreshold), 0.5 J led to significant changes for one or more phenotypes (threshold), and 0.6 and 0.7 J resulted in significant changes in all outcomes assessed (mTBI). We further show that linear head kinematics are the most robust predictors of duration of unconsciousness, severity of neurological deficits, white matter injury, and microgliosis following single TBI. Our data extend the validation of CHIMERA as a biofidelic animal model of DAI and establish working parameters to guide future investigations of the mechanisms underlying axonal pathology and inflammation induced by mechanical trauma. HIGHLIGHTSBiomechanical input energy predicts biological responses in mouse CHIMERA TBI.Impact energies of 0.4 J and below are subthreshold and produce no injury phenotype.Injury threshold is 0.5 J, where at least one biological outcome is altered.Mild TBI phenotypes are observed at 0.6 J and above.