Altered neurotransmission in the lateral amygdala in aged human apoE4 targeted replacement mice

The human APOE4 allele is associated with an early age of onset and increased risk of Alzheimer's disease (AD). Apolipoprotein E is secreted as part of a high-density lipoprotein-like particle by glial cells in the brain for the primary purpose of transport of lipophilic compounds involved in the maintenance of synapses. Previous studies examining synaptic integrity in the amygdala of human apoE targeted replacement (TR) mice showed a decrease in spontaneous excitatory synaptic activity, dendritic arbor, and spine density associated with apoE4 compared with apoE3 and apoE2 in adult male mice. In the present study, we assessed how APOE genotype affects synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in human apoE3, E4, and E2/4 TR mice at the age of 18–20 months. In contrast to adult mice, we found that aged apoE4 TR mice exhibited the highest level of excitatory synaptic activity compared with other cohorts. Additionally, apoE4 mice had significantly greater spontaneous inhibitory activity than all other cohorts. Taken together, there was a significant interaction between genotypes when comparing inhibition relative to excitation; there was a simple main effect of frequency type with an imbalance toward inhibition in apoE4 mice but not in apoE3 or apoE2/4 mice. These results suggest that apoE isoforms differentially influence synaptic transmission throughout the life span, where aging coupled with apoE4 expression, results in an imbalance in maintaining integrity of synaptic transmission.     

An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.     

Apolipoprotein E receptors and amyloid expression are modulated in an apolipoprotein E-dependent fashion in response to hippocampal deafferentation in rodent

The entorhinal cortex lesion paradigm is a widely accepted and efficient method to provoke reactive synaptogenesis and terminal remodeling in the adult CNS. This approach has been used successfully to contrast the profile of reactivity from various proteins associated with Alzheimer's disease pathophysiology in wild-type and apolipoprotein E (apoE)-deficient (APOE ko) mice. Results indicate that the production of the beta-amyloid 1-40 peptide (A beta 40) is increased in response to neuronal injury, with a timing that is different between wild-type and APOE ko animals. Moreover, we report that baseline levels of the A beta 40 peptide are significantly higher in the APOE ko mice. The expression of the apolipoprotein E receptor type 2 (apoER2) is also modulated by the deafferentation process in the hippocampus, but only in APOE ko mice. These results provide novel insights as to the molecular mechanisms responsible for the poor plastic response reported in apoE4-expressing and apoE deficient mice in response to hippocampal injury.     

Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer's disease patients

Dendritic spines are postsynaptic sites of excitatory input in the mammalian nervous system. Apolipoprotein (apo) E participates in the transport of plasma lipids and in the redistribution of lipids among cells. A role for apoE is implicated in regeneration of synaptic circuitry after neural injury. The apoE4 allele is a major risk factor for late-onset familial and sporadic Alzheimer's disease (AD) and is associated with a poor outcome after brain injury. ApoE isoforms are suggested to have differential effects on neuronal repair mechanisms. In vitro studies have demonstrated the neurotrophic properties of apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype on neuronal cell dendritic spine density in mice and in human postmortem tissue. In order to compare the morphology of neurons developing under different apoE conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG) granule cells of the hippocampus were carried out in wild-type (WT), human apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice; the same dendritic spine parameters were also assessed in human postmortem DG from individuals with and without the apoE4 gene. Quantitative analysis of dendritic spine length, morphology, and number was carried out on these mice at 3 weeks, 1 and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001), while at 3 weeks there were no differences between the groups. These age dependent differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of dementia in aged individuals with the apoE4 allele. Significantly in human brain, apoE4 dose correlated inversely with dendritic spine density of DG neurons cell in the hippocampus of both AD (P=0.0008) and aged normal controls (P=0.0015). Our findings provide one potential explanation for the increased cognitive decline seen in aged and AD patients expressing apoE4.     

Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice

Background  

Inheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid β metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the ε4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity.     

Influence of the apolipoprotein E epsilon4 allele on human embryonic development

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.     

Genotype-related differences of hippocampal apolipoprotein E levels only in early stages of neuropathological changes in Alzheimer's disease

Inheritance of the epsilon4 allele of apolipoprotein E (APOE, gene; apoE, protein) represents the most common genetic risk factor for developing Alzheimer's disease (AD), but the role of apoE in AD pathogenesis is yet to be clarified. A number of studies investigating apoE expression and protein levels in AD brain in correlation to its genetic polymorphism has yielded controversial results. We designed our approach based on neuropathological characteristics of AD to investigate apoE levels in relation to the APOE genotype and AD-related neurofibrillary changes, and amyloid deposits. We determined hippocampal apoE levels by reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting in 70 Braak-staged and APOE-genotyped autopsy brains. In our stage-, age- and gender-matched case sample, we found a significant increase of hippocampal apoE in the APOE epsilon3 homozygotes with beginning AD-related pathology (Braak stages I and II) compared with brain samples free of neurofibrillary changes and amyloid deposits. In the APOE epsilon4 allele carriers no such increase was found. In both genotype groups, severely affected brain samples with widespread neurofibrillary changes (Braak stages V and VI) and amyloid deposits (Braak stage C) showed low apoE levels comparable to those found in unaffected brain samples (Braak stage 0).Our data suggests that the isoform-specific impact of apoE on the development of AD may be of crucial importance only in the early stages of the disease. These stages are believed to represent phases of the disease in which the beginning neurodegeneration can be compensated by plastic reorganization.     

Acetylcholine receptor and behavioral deficits in mice lacking apolipoprotein E

Apolipoprotein E (apoE) is involved in the risk to develop sporadic Alzheimer's disease (AD). Since impaired central acetylcholine (ACh) function is a hallmark of AD, apoE may influence ACh function by modulating muscarinic ACh receptors (mAChRs). To test this hypothesis, mAChR binding was measured in mice lacking apoE and wild type C57BL/6J mice. Mice were also tested on the pre-pulse inhibition, delay eyeblink classical conditioning, and 5-choice serial reaction time tasks (5-SRTT), which are all modulated by ACh transmission. Mice were also given scopolamine to challenge central mAChR function. Compared to wild type mice, mice lacking apoE had reduced number of cortical and hippocampal mAChRs. Scopolamine had a small effect on delay eyeblink classical conditioning in wild type mice but a large effect in mice lacking apoE. Mice lacking apoE were also unable to acquire performance on the 5-SRTT. These results support a role for apoE in ACh function and suggest that modulation of cortical and hippocampal mAChRs might contribute to genotype differences in scopolamine sensitivity and task acquisition. Impaired apoE functioning may result in cholinergic deficits that contribute to the cognitive impairments seen in AD.     

Comparison of astrocytic and myocytic metabolic dysregulation in apolipoprotein E deficient and human apolipoprotein E transgenic mice

The accumulation of tubular aggregates in type II skeletal muscle fibres and fibrillo-granular inclusions in hippocampal protoplasmic astrocytes are characteristic lesions of apolipoprotein E deficient mice. Moreover these inclusions reacted immunocytochemically with an antibody specific to fragment 17-24 of the published sequence of Alzheimer's amyloid peptide. In an effort to evaluate the role of apolipoprotein E in the formation of these abnormal structures, we examined the tibialis anterior muscle and the hippocampus of several groups of animals including: (i) apolipoprotein E "knockout" mice which had been whole body irradiated with 1200 rads and bone marrow replenished with apolipoprotein E sufficient marrow; and (ii) three transgenic murine strains that had been genetically engineered to express either human apolipoprotein E2, E3 or E4 protein on an apoE deficient background. The results of this study showed that the presence of murine apolipoprotein E (even in subnormal levels in the serum) in irradiated bone marrow replenished mice and in all three (E2, E3 or E4) human apoE transgenic strains was sufficient to prevent the aggregation of sarcoplasmic tubules in the tibialis anterior type II muscle fibres. Similarly apolipoprotein E "knockout" bone marrow replenished mice and all three transgenic strains expressing the different human apolipoprotein E alleles reduced the number of the astrocytic inclusions in the hippocampus to levels not significantly different to those observed in control C57Bl6J animals.The data obtained in this study indicate that neurological and neuromuscular abnormalities found in apoE deficient mice are reversed when apoE protein is replaced in the circulation, either by bone marrow transplantation of normal apoE sufficient marrow, or by gene therapy with the apoE gene, albeit of human origin and irrespective of the allele used.     

Altered cholesterol metabolism in human apolipoprotein E4 knock-in mice

Thevarepsilon4 allele of apolipoprotein E (apoE) is associated with an increased risk of developing Alzheimer's disease (AD). To accurately determine the isoform-specific effects of human apoE on brain functions under physiological and pathological situations, we created mice expressing human apoE4 isoform in place of mouse apoE by utilizing the gene-targeting technique on the embryonic stem cells (knock-in). The homozygousvarepsilon4 (4/4) mice correctly expressed human apoE4 in the serum and the brain. The human apoE in the brain was found primarily in the astrocytes as was the mouse apoE in the wild-type (+/+) mice. In the 4/4 mice, the serum cholesterol level was 2.5-fold that of the +/+ littermate controls on a regular diet. This marked elevation was accounted for by an accumulation of very low and low density lipo-proteins. In the brains of the 4/4 mice, however, the amounts of total cholesterol and phospholipids were significantly decreased compared with the +/+ littermates. These findings indicate that cholesterol and lipid metabolism is markedly altered in the 4/4 mice. Our human apoE4 knock-in mice will be useful in clarifying the role of apoE in the etiologies of AD and cardiovascular diseases in relation to cholesterol and lipid metabolism.     

Distortion of allelic expression of apolipoprotein E in Alzheimer's disease

The APOE epsilon4 allele is a strong genetic susceptibility factor for Alzheimer's disease. Interaction with other biological factors may modulate the effect of the apoE isoforms. However, previous work suggested that other genetic variability within the APOE locus, influencing the effect of the epsilon4 allele, may exist. Such variability could modify the expression of the APOE gene and, in particular, the level of expression of APOE alleles could be an important determinant of disease pathogenesis. To test this hypothesis we examined the levels of expression of APOE in heterozygotes with AD and in controls, using a new method of semi-quantitation. We report that relative epsilon4 mRNA expression is increased in AD compared with controls and suggest that genetic variability in the neural expression of APOE contributes to disease risk.      

Alteration of nitric oxide synthase activity in young and aged apolipoprotein E-deficient mice

Impairments in cognitive performance have been observed in aged apolipoprotein E (apoE)-deficient mice, and apoE epsilon 4 allele is a risk factor in Alzheimer's disease (AD). The absence of apoE correlates with diminished antioxidative capacity in animals, and elevated cerebral oxidative stress has been observed in AD individuals carrying the epsilon 4 alleles. Nitric oxide (NO) is a neurosignaling molecule that has significant roles in cognition. NO has also been implicated in neurodegenerative diseases due to its oxidative properties. The current study examined the possible relationship between apoE and nitric oxide synthase (NOS) by comparing hippocampal and cortical NOS activities in wild-type and apoE-knockout mice. Our results showed that apoE deficiency had no effect on NOS activity in these animals; however, aged animals uniformly exhibited significantly higher NOS activity levels. These findings suggest that increased NOS activity may contribute to cognitive impairments in aged wild-type and apoE-knockout mice due to excess accumulation of oxidative damages in areas involved in learning and memory.     

Reduced levels of human apoE4 protein in an animal model of cognitive impairment

The APOE4 allele is the most common genetic determinant for Alzheimer's disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4's link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.     

Dominant negative effects of apolipoprotein E4 revealed in transgenic models of neurodegenerative disease

Apolipoprotein E fulfills fundamental functions in lipid transport and neural tissue repair after injury.(6,8) Its three most common isoforms (E2, E3, and E4) are critical determinants of diverse human diseases, including major cardiovascular and neurodegenerative disorders.(8,14) Apolipoprotein E4 is associated with an increased risk for Alzheimer's disease(3,5) and poor clinical outcome after head injury or stroke.(11,16) The precise role of apolipoprotein E4 in these conditions remains unknown. To characterize the effects of human apolipoprotein E isoforms in vivo, we analysed transgenic Apoe knockout mice that express apolipoprotein E3 or E4 or both in the brain. Hemizygous and homozygous apolipoprotein E3 mice were protected against age-related and excitotoxin-induced neurodegeneration, whereas apolipoprotein E4 mice were not. Apolipoprotein E3/E4 bigenic mice were as susceptible to neurodegeneration as apolipoprotein E4 singly-transgenic mice. At eight months of age neurodegeneration was more severe in homozygous than in hemizygous apolipoprotein E4 mice consistent with a dose effect. Thus, apolipoprotein E4 is not only less neuroprotective than apolipoprotein E3 but also acts as a dominant negative factor that interferes with the beneficial function of apolipoprotein E3. The inhibition of this apolipoprotein E4 activity may be critical for the prevention and treatment of neurodegeneration in APOE varepsilon4 carriers.     

Apolipoprotein E isoform-dependent dendritic recovery of hippocampal neurons following activation of innate immunity

Background  

Innate immune activation, including a role for cluster of differentiation 14/toll-like receptor 4 co-receptors (CD14/TLR-4) co-receptors, has been implicated in paracrine damage to neurons in several neurodegenerative diseases that also display stratification of risk or clinical outcome with the common alleles of the apolipoprotein E gene (APOE): APOE2, APOE3, and APOE4. Previously, we have shown that specific stimulation of CD14/TLR-4 with lipopolysaccharide (LPS) leads to greatest innate immune response by primary microglial cultures from targeted replacement (TR) APOE4 mice and greatest p38MAPK-dependent paracrine damage to neurons in mixed primary cultures and hippocampal slice cultures derived from TR APOE4 mice. In contrast, TR APOE2 astrocytes had the highest NF-kappaB activity and no neurotoxicity. Here we tested the hypothesis that direct activation of CD14/TLR-4 in vivo would yield different amounts of paracrine damage to hippocampal sector CA1 pyramidal neurons in TR APOE mice.     

APOE and the regulation of microglial nitric oxide production: a link between genetic risk and oxidative stress

The mechanism linking the APOE4 gene with increased susceptibility for Alzheimer's disease (AD) and poorer outcomes following closed head injury and stroke is unknown. One potential link is activation of the innate immune system in the CNS. Our previously published data demonstrated that apolipoprotein E regulates production of nitric oxide, a critical cytoactive factor released by immune active macrophages. To determine if immune regulation is different in the presence of apolipoprotein E4 compared to apolipoprotein E3, we have measured NO production by peritoneal and CNS macrophages (microglia) cultured from transgenic mice that only express the human apoE4 or apoE3 protein isoform. Significantly more NO was produced in APOE4 mice compared to APOE3 transgenic mice that only express human apoE3 protein. Similarly, monocyte derived macrophages from humans carrying APOE4 gene alleles also produce significantly greater NO than those individuals with APOE3. The mechanism for this isoform-specific difference in NO production is not known and multiple sites in the NO production pathway may be affected. Expression of inducible nitric oxide synthase (iNOS) mRNA and protein are not significantly different between the APOE3 and APOE4 mice, suggesting that induction of iNOS is not a primary cause of the increased NO production in APOE4 animals. One alternative regulatory mechanism that demonstrates isoform specificity is arginine transport, which is greater in microglia from APOE4 transgenic mice compared to microglia from APOE3 mice. Increased transport is consistent with an increased production of NO and may reflect a direct or indirect effect of the APOE genotype on microglial arginine uptake and microglial activation in general. Overall, greater NO production in APOE4 carriers where characteristically high levels of oxidative/nitrosative stress are found in diseases such as AD provides a mechanism that potentially explains the genetic association between APOE4 and human diseases.     

Marked regional differences of brain human apolipoprotein E expression in targeted replacement mice

We used three human apolipoprotein (apo) E targeted replacement mouse lines, each expressing one of the three common human apoE isoforms to study the pattern of apoE expression in the central nervous system (CNS). Immunocytochemistry on brain sections from all three lines of targeted replacement mice, wild type mice, African green monkeys, and humans show a predominantly glial pattern of apoE expression. The levels of human apoE protein in hippocampus and frontal cortex were similar between targeted replacement mice and non-demented human tissue. Within a given brain region, the levels of apoE were very similar amongst all three isoforms, which contrasts sharply with plasma, where apoE2 levels are 16-fold higher than apoE3 and E4 levels. Across brain regions, cerebellar apoE levels were significantly higher than cerebral apoE levels. In conclusion, we provide detailed analysis of a human apoE animal model system that recapitulates both the pattern and level of apoE expression in non-demented humans. The neurobiology of human apoE isoforms can now be studied in both the normal and post-injury state, since all apoE regulatory sequences are intact. Finally, the differences in apoE levels we observed may explain the regional vulnerability of neuronal degeneration in Alzheimer's disease.     

The role of apolipoprotein E in Alzheimer's disease, acute brain injury and cerebrovascular disease: evidence of common mechanisms and utility of animal models

The epsilon 4 allele of apolipoprotein E (APOE denotes gene; apoE denotes protein) is a major risk factor for Alzheimer's disease (AD). More recent evidence indicates an association with a poor outcome after acute brain injury including that due to head trauma and intracerebral hemorrhage. APOE gene polymorphism also influences the risk of hemorrhage in cerebral amyloid angiopathy. These diverse brain disorders seem to have some mechanisms in common. The multiplicity of the roles of apoE within the central nervous system is currently being unraveled. For example, apoE can interact with amyloid beta-protein and tau, proteins central to the pathogenesis of AD. In addition to these effects, it is proposed that one of the major functions of apoE is to mediate neuronal protection, repair and remodeling. In all of the different roles proposed, there are marked apoE-isoform specific differences. Although it remains to be clarified which is the most important mechanism(s) in each disorder in which apoE is involved, these isoform specific differences seem to underly a genetically determined susceptibility to outcome from acute brain injury and to AD with APOE epsilon 4 conferring relative vulnerability. This review focuses on apoE research, from clinical studies to animal models, in AD, acute brain injury and cerebrovascular disease and explores the common mechanisms that may explain some of the complex underlying neurobiology.     

Apolipoprotein E4 decreases whereas apolipoprotein E3 increases the level of secreted amyloid precursor protein after closed head injury

Apolipoprotein E (apoE4) and head trauma are important genetic and environmental risk factors for Alzheimer's disease. Furthermore, apoE4 increases both the acute and chronic consequences of head trauma. The latter are associated with the deposition of amyloid-beta, which is particularly elevated in apoE4 subjects. The short-term effects of head injury are associated with transiently increased metabolism of amyloid precursor protein (APP) and its secreted fragment, APPs. In the present study, we examined the possibility that the acute, short-term pathological effects of apoE4 following head trauma and the corresponding neuroprotective effects of apoE3 are related to isoform-specific effects of apoE on APP metabolism. Accordingly, male transgenic mice expressing human apoE3 or apoE4 on a null mouse apoE background and apoE-deficient and control mice were subjected to closed head injury (CHI). The resulting effects on brain APP, and on its secreted products, APPs and secreted product of the alpha-cleavage of APP (APPsalpha) were then determined 24 h following injury. Immunoblotting revealed no significant differences between the basal APP, APPs and APPsalpha levels of the hippocampus or the cortex of the control and the apoE3 and ApoE4 transgenic mice. The apoE-deficient mice also had similar cortical basal levels of APP and its metabolites, whereas their corresponding basal hippocampal APP and APPs levels were lower than those of the other groups. CHI lowered the hipppocampal APPs and APPsalpha levels of the apoE4 transgenic mice, whereas those of the apoE3 transgenic mice and of the control and apoE-deficient mice were not affected by this insult. In contrast, CHI raised the cortical APP and APPs levels of the apoE3 transgenic mice but had no significant effect on those of the other mice groups. These animal model findings suggest that the acute, short-term pathological effects of apoE4 following CHI and the corresponding neuroprotective effects of apoE3 may be mediated by their opposing effects on the expression and cleavage of cortical and hippocampal APP. Similar isoform-specific interactions between apoE and APP may play a role in the acute, short-term effects of head trauma in humans.