Courtney Lane-Donovan

Reelin protects against amyloid β toxicity in vivo

Reelin prevents the deleterious effects of amyloid β on synaptic transmission, learning, and memory. Protecting neurons from amyloid β In the developing nervous system, the secreted protein Reelin helps to guide migrating neurons to their correct destination. In the adult nervous system, Reelin enhances synaptic plasticity and protects isolated neurons from the toxicity of amyloid β, the accumulation of which causes the neurodegeneration characteristic of Alzheimer’s disease. To avoid the developmental defects associated with Reelin deficiency, Lane-Donovan et al. generated mice with an inducible knockout of Reelin that accumulated amyloid β. Mice that lacked Reelin as adults showed greater defects in synaptic plasticity, learning, and memory in response to amyloid β accumulation, indicating that Reelin protects against the neurotoxicity of amyloid β in vivo. Alzheimer’s disease (AD) is a currently incurable neurodegenerative disorder and is the most common form of dementia in people over the age of 65 years. The predominant genetic risk factor for AD is the ε4 allele encoding apolipoprotein E (ApoE4). The secreted glycoprotein Reelin enhances synaptic plasticity by binding to the multifunctional ApoE receptors apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr). We have previously shown that the presence of ApoE4 renders neurons unresponsive to Reelin by impairing the recycling of the receptors, thereby decreasing its protective effects against amyloid β (Aβ) oligomer–induced synaptic toxicity in vitro. We showed that when Reelin was knocked out in adult mice, these mice behaved normally without overt learning or memory deficits. However, they were strikingly sensitive to amyloid-induced synaptic suppression and had profound memory and learning disabilities with very low amounts of amyloid deposition. Our findings highlight the physiological importance of Reelin in protecting the brain against Aβ-induced synaptic dysfunction and memory impairment.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning

Glycosylation of the apolipoprotein E receptor Apoer2 is important for regulating synaptic function and cognition. Sugar for Normal Brain Function Alzheimer’s disease is a neurodegenerative disorder that results in dementia. Decreased signaling through the receptor Apoer2 exacerbates some of the molecular changes that occur in Alzheimer’s disease. Wasser et al. generated mice with a form of Apoer2 lacking the domain that is heavily glycosylated with O-linked sugars. The abundance of this mutant receptor in these mice was higher than that of Apoer2 in wild-type mice. Lack of this domain resulted in changes in synaptic morphology and composition, decreased synaptic efficacy, and defects in learning and memory. These neurological effects appeared to depend on the increased amount of the mutant receptor because they were absent in mice with lower amounts of the mutant receptor. Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase–dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory.

ApoE, ApoE Receptors, and the Synapse in Alzheimer's Disease

As the population ages, neurodegenerative diseases such as Alzheimers disease (AD) are becoming a significant burden on patients, their families, and health-care systems. Neurodegenerative processes may start up to 15 years before outward signs and symptoms of AD, as evidenced by data from AD patients and mouse models. A major genetic risk factor for late-onset AD is the ɛ4 isoform of apolipoprotein E (ApoE4), which is present in almost 20% of the population. In this review we discuss the contribution of ApoE receptor signaling to the function of each component of the tripartite synapse - the axon terminal, the postsynaptic dendritic spine, and the astrocyte - and examine how these systems fail in the context of ApoE4 and AD.

Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice

Alzheimers disease (AD) is the most common form of dementia in individuals over the age of 65 years. The most prevalent genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are being considered. One unresolved question in ApoE biology is whether ApoE is necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic loss and cognitive dysfunction; however, these findings are complicated by the fact that ApoE knock-out mice have highly elevated plasma lipid levels, which may independently affect brain function. To bypass the effect of ApoE loss on plasma lipids, we generated a novel mouse model that expresses ApoE normally in peripheral tissues, but has severely reduced ApoE in the brain, allowing us to study brain ApoE loss in the context of a normal plasma lipid profile. We found that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction similar to that of ApoE KO mice; however, the bEKO mice did not have the learning and memory impairment observed in ApoE KO mice. Moreover, we found that the memory deficit in the ApoE KO mice was specific to female mice and was fully rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These findings suggest that plasma lipid levels can influence cognition and synaptic function independent of ApoE expression in the brain. SIGNIFICANCE STATEMENT One proposed treatment strategy for Alzheimers disease (AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk factor for the disease. A major concern of this strategy is that an animal model of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and cognitive impairment; however, these mice also develop dyslipidemia and severe atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to wild-type levels normalizes plasma lipids in ApoE KO mice. While this does not rescue synaptic loss, it does completely restore learning and memory in the mice, suggesting that both CNS and plasma ApoE are independent parameters that affect brain health.

Lrp4 Domains Differentially Regulate Limb/Brain Development and Synaptic Plasticity

Apolipoprotein E (ApoE) genotype is the strongest predictor of Alzheimer’s Disease (AD) risk. ApoE is a cholesterol transport protein that binds to members of the Low-Density Lipoprotein (LDL) Receptor family, which includes LDL Receptor Related Protein 4 (Lrp4). Lrp4, together with one of its ligands Agrin and its co-receptors Muscle Specific Kinase (MuSK) and Amyloid Precursor Protein (APP), regulates neuromuscular junction (NMJ) formation. All four proteins are also expressed in the adult brain, and APP, MuSK, and Agrin are required for normal synapse function in the CNS. Here, we show that Lrp4 is also required for normal hippocampal plasticity. In contrast to the closely related Lrp8/Apoer2, the intracellular domain of Lrp4 does not appear to be necessary for normal expression and maintenance of long-term potentiation at central synapses or for the formation and maintenance of peripheral NMJs. However, it does play a role in limb development.

The ApoE receptors Vldlr and Apoer2 in Central Nervous System Function and Disease

The LDL receptor (LDLR) family has long been studied for its role in cholesterol transport and metabolism; however, the identification of ApoE4, an LDLR ligand, as a genetic risk factor for late-onset Alzheimer’s disease has focused attention on the role this receptor family plays in the CNS. Surprisingly, it was discovered that two LDLR family members, ApoE receptor 2 (Apoer2) and VLDL receptor (Vldlr), play key roles in brain development and adult synaptic plasticity, primarily by mediating Reelin signaling. This review focuses on Apoer2 and Vldlr signaling in the CNS and its role in human disease.

High-Fat Diet Changes Hippocampal Apolipoprotein E (ApoE) in a Genotype- and Carbohydrate-Dependent Manner in Mice

Alzheimer’s disease is a currently incurable neurodegenerative disease affecting millions of individuals worldwide. Risk factors for Alzheimer’s disease include genetic risk factors, such as possession of ε4 allele of apolipoprotein E (ApoE4) over the risk-neutral ApoE3 allele, and lifestyle risk factors, such as diet and exercise. The intersection of these two sources of disease risk is not well understood. We investigated the impact of diet on ApoE levels by feeding wildtype, ApoE3, and ApoE4 targeted replacement (TR) mice with chow, high-fat, or ketogenic (high-fat, very-low-carbohydrate) diets. We found that high-fat diet affected both plasma and hippocampal levels of ApoE in an isoform-dependent manner, with high-fat diet causing a surprising reduction of hippocampal ApoE levels in ApoE3 TR mice. Conversely, the ketogenic diet had no effect on hippocampal ApoE. Our findings suggest that the use of dietary interventions to slow the progression AD should take ApoE genotype into consideration.

Is Apolipoprotein E Required for Cognitive Function in Humans?: Implications for Alzheimer Drug Development

More than twenty years ago, a polymorphism in the Apolipoprotein E (apoE) gene was identified as the primary risk factor for late-onset Alzheimer disease (AD).1 Individuals carrying the ɛ4 isoform of apoE (apoE4) are at significantly greater risk for AD compared to apoE3 carriers, whereas the apoE2 allele is associated with reduced AD risk.2 Despite two decades of research into the mechanisms by which apoE4 contributes to disease pathogenesis, a seemingly simple question remains unresolved: Is apoE good or bad for brain health? The answer to this question is essential for the future development of apoE-directed therapeutics. In JAMA Neurology, Mak et al. describe a patient who has undetectable levels of apoE in plasma and cerebrospinal fluid.3 The patient, a 40-year-old African American man, is homozygous for a rare loss-of-function mutation in apoE. Despite severe dysbetalipoproteinemia and xanthomatosis, the patient is cognitively normal and shows no signs of neurodegeneration. In light of apoE as the primary risk factor for AD, the lack of neurological findings in this patient would appear to answer the question of whether apoE is necessary for brain function with a resounding no. In the plasma, apoE is a carrier of chylomicron remnants and very-low-density lipoprotein particles and binds to members of the low-density lipoprotein receptor family for uptake into cells. These lipoprotein particles cannot cross the blood-brain barrier; thus, apoE-containing particles released by astrocytes are the main source of brain apoE.4,5 The importance of circulating apoE is highlighted by the greatly elevated total cholesterol levels, contained mostly in the very low-density lipoprotein fraction, in the apoE-deficient patient described by Mak and colleagues, leading to prominent xanthomatosis3 and, as had been reported in other rare cases of apoE deficiency, a concomitant increase in cardiovascular disease risk.6 However, the previous case studies did not investigate potential neurological sequelae of apoE deficiency. Several mouse apoE knockouts have been generated to investigate the effect of apoE loss on atherosclerosis and cognition, sometimes with conflicting results. Consistently, apoE knockout mice have elevated cholesterol levels, similar to apoE-deficient patients.7 Neurological findings in these mice have been less clear, however. Some groups have reported that while apoE knockout mice develop normally, they begin to lose neurons around 5 months of age accompanied by a reduction in working memory,8,9 which is ameliorated by infusion with recombinant apoE3 or apoE4.10 These findings have been challenged by other studies that found no age-related neurodegeneration in apoE knockout mice.11,12 Alzheimer disease is characterized by the progressive buildup of neuritic plaques comprising fibrillar amyloid beta (Aβ; a cleavage product of the amyloid precursor protein), followed by the appearance of neurofibrillary tangles of hyperphosphorylated tau. These toxic particles affect a variety of processes, including inflammation, synaptic plasticity, and cell viability.13,14 Apolipoprotein E4 promotes disease pathogenesis through both Aβ-dependent and Aβ-independent pathways. Carriers of apoE4 have earlier and more rapid deposition of plaques, which is thought to stem from impaired clearance of Aβ from the interstitial fluid15,16 and from fibrillogenesis-promoting properties of apoE4.17 Also, apoE4 modifies brain function independent of Aβ, as apoE4 carriers show altered connectivity patterns by magnetic resonance imaging in AD-related brain regions as early as infancy.18 Because murine apoE differs from the 3 human apoE isoforms at numerous amino acid residues, human apoE knock-in mice have been developed to study the roles of the isoforms in vivo in the mouse. These apoE knock-in mice have been crossed with mouse models of AD that carry familial AD mutations in APP or PSEN1, which cause high levels of Aβ and age-related plaque deposition and cognitive decline. Consistent with findings in human populations, the presence of apoE4 accelerates the development of plaques and cognitive dysfunction in AD mice over apoE3 and apoE2.19 Intriguingly, gene transfer by adeno-associated virus of the 3 human apoE isoforms into AD mice on a normal mouse apoE background showed that while apoE4 increased plaques, apoE2 actually decelerated plaque growth. However, adeno-associated virus transfer of human apoE also reduced endogenous murine apoE levels by approximately 15%.20 It is important to point out in this context that the reduction of endogenous mouse apoE alone – or its replacement with any human apoE allele – by itself reduces plaque deposition and ameliorates cognitive deficits in the animals.21,22 Several apoE-directed AD therapeutics are currently in development. Some seek to increase apoE levels and lipidation by upregulating expression of adenosince triphosphate-binding cassette transporter A1 protein (ABCA1), eg, through liver X receptor and retinoid X receptor agonists.23,24 Conversely, other approaches aim at lowering apoE levels. Specifically, passive immunotherapy with anti-apoE antibodies is currently being tested in animal models and has been shown to reduce Aβ deposition in mouse models of AD.25 One caveat for the development of systemic apoE-directed therapeutics is the effect of apoE loss on plasma lipoproteins. Both the patient described by Mak and colleagues and apoE knockout mice have impaired clearance of lipoproteins, resulting in massively elevated cholesterol levels in the circulation that could lead to accelerated atherosclerosis3. To prevent the disruption of lipid metabolism in AD patients, apoE-directed drugs will need to be brain-specific. This could take the form of specifically targeting the drug to the central nervous system or targeting apoE-specific interactions in the central nervous system (eg, Aβ binding). Promisingly, systemic administration of anti-apoE antibodies does not alter cholesterol levels in a mouse model of AD, while still modestly reducing levels of Aβ and improving cognition.25 The grossly normal cognitive status of the apoE-deficient patient described by Mak and colleagues suggests that therapeutics that reduce cerebral apoE levels will likely not adversely affect cognition in at-risk patients. Because the described patient is only 40 years old, close clinical follow-up is needed to determine if he is at increased risk for age-dependent cognitive decline; however, his grossly elevated lipid profile may confound this because increased plasma cholesterol is independently associated with increased AD risk.26 Overall, the patient’s normal cognitive function together with the earlier mouse work suggest that interventions that reduce cerebral apoE levels may hold promise as a potential therapeutic approach to AD.

Building a better blood-brain barrier

A new three-dimensional model of the blood-brain barrier can be used to study processes that are involved in neurodegenerative diseases.

NGP 555, a γ-secretase modulator, lowers the amyloid biomarker, Aβ42, in cerebrospinal fluid while preventing Alzheimer's disease cognitive decline in rodents

Alzheimers disease (AD) is defined by the progressive accumulation of amyloid plaques and neurofibrillary tangles in the brain which precedes cognitive decline by years.