Haim Belinson

Activation of the Amyloid Cascade in Apolipoprotein E4 Transgenic Mice Induces Lysosomal Activation and Neurodegeneration Resulting in Marked Cognitive Deficits

The allele E4 of apolipoprotein E (apoE4), the most prevalent genetic risk factor for Alzheimers disease, is associated histopathologically with elevated levels of brain amyloid. This led to the suggestion that the pathological effects of apoE4 are mediated by cross-talk interactions with amyloid β peptide (Aβ), which accentuate the pathological effects of the amyloid cascade. The mechanisms underlying the Aβ-mediated pathological effects of apoE4 are unknown. We have shown recently that inhibition of the Aβ-degrading enzyme neprilysin in brains of wild-type apoE3 and apoE4 mice results in rapid and similar elevations in their total brain Aβ levels. However, the nucleation and aggregation of Aβ in these mice were markedly affected by the apoE genotype and were specifically enhanced in the apoE4 mice. We presently used the neprilysin inhibition paradigm to analyze the neuropathological and cognitive effects that are induced by apoE4 after activation of the amyloid cascade. This revealed that apoE4 stimulates isoform specifically the degeneration of hippocampal CA1 neurons and of entorhinal and septal neurons, which is accompanied by the accumulation of intracellular Aβ and apoE and with lysosomal activation. Furthermore, these neuropathological effects are associated isoform specifically with the occurrence of pronounced cognitive deficits in the ApoE4 mice. These findings provide the first in vivo evidence regarding the cellular mechanisms underlying the pathological cross talk between apoE4 and Aβ, as well as a novel model system of neurodegeneration in vivo that is uniquely suitable for studying the early stages of the amyloid cascade and the effects thereon of apoE4.

ApoE4-dependent Aβ-mediated neurodegeneration is associated with inflammatory activation in the hippocampus but not the septum

Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimer’s disease, is histopathologically associated with increased deposition of amyloid-β and brain inflammation and with impaired neuronal plasticity and repair. We have recently shown that the activation of the amyloid cascade by inhibition of the Aβ-degrading enzyme, neprilysin, stimulates the isoform-specific degeneration of hippocampal CA1 neurons and septal neurons in apoE4 transgenic mice and that this effect is accompanied by the accumulation of intracellular Aβ in the affected neurons. We presently examined the extent to which this apoE4-dependent Aβ-mediated neurodegeneration is associated with brain area specific inflammatory activation. This revealed that the activation of the amyloid cascade in apoE transgenic mice results in the activation of microgliosis and astrogliosis in the hippocampus of apoE4, but not in apoE3 transgenic mice. The effect was most pronounced in the hippocampal CA1 subfield and its initial kinetics followed that of the accumulation of Aβ in CA1 neurons. In contrast, the corresponding apoE4-dependent Aβ degeneration of septal neurons was not associated with the activation of either gliosis or astrogliosis in this brain area. These animal model findings, that the association between brain inflammation and neurodegeneration is brain area specific, suggest that neuropathological inflammatory interactions in AD may also be brain area specific and that consequently the efficacy of putative anti-inflammatory intervention may also be brain area selective.

Intraneuronal amyloid‐β plays a role in mediating the synergistic pathological effects of apoE4 and environmental stimulation

The allele E4 of apolipoprotein E4 (apoE4), which is the most prevalent genetic risk factor of Alzheimer’s disease (AD), inhibits synaptogenesis and neurogenesis and stimulates apoptosis in brains of apoE4 transgenic mice that have been exposed to an enriched environment. In the present study, we investigated the hypothesis that the brain activity‐dependent impairments in neuronal plasticity, induced by apoE4, are mediated via the amyloid cascade. Importantly, we found that exposure of mice transgenic for either apoE4, or the Alzheimer’s disease benign allele apoE3, to an enriched environment elevates similarly the hippocampal levels of amyloid‐β peptide (Aβ) and apoE of these mice, but that the degree of aggregation and spatial distribution of Aβ in these mice are markedly affected by the apoE genotype. Accordingly, environmental stimulation triggered the formation of extracellular plaque‐like Aβ deposits and the accumulation of intra‐neuronal oligomerized Aβ specifically in brains of apoE4 mice. Further experiments revealed that hippocampal dentate gyrus neurons are particularly susceptible to apoE4 and environmental stimulation and that these neurons are specifically enriched in both oligomerized Aβ and apoE. These findings show that the impairments in neuroplasticity which are induced by apoE4 following environmental stimulation are associated with the accumulation of intraneuronal Aβ and suggest that oligomerized Aβ mediates the synergistic pathological effects of apoE4 and environmental stimulation.

ApoE4-Driven Accumulation of Intraneuronal Oligomerized Aβ42 following Activation of the Amyloid Cascade In Vivo Is Mediated by a Gain of Function

Activating the amyloid cascade by inhibiting the Aβ-degrading enzyme neprilysin in targeted replacement mice, which express either apoE4 or apoE3, results in the specific accumulation of oligomerized Aβ42 in hippocampal CA1 neurons of the apoE4 mice. We presently investigated the extent to which the apoE4-driven accumulation of Aβ42 and the resulting mitochondrial pathology are due to either gain or loss of function. This revealed that inhibition of neprilysin for one week triggers the accumulation of Aβ42 in hippocampal CA1 neurons of the apoE4 mice but not of either the corresponding apoE3 mice or apoE-deficient mice. At 10 days, Aβ42 also accumulated in the CA1 neurons of the apoE-deficient mice but not in those of the apoE3 mice. Mitochondrial pathology, which in the apoE4 mice is an early pathological consequence following inhibition of neprilyisn, also occurs in the apoE-deficient but not in the apoE3 mice and the magnitude of this effect correlates with the levels of accumulated Aβ42 and oligomerized Aβ42 in these mice. These findings suggest that the rate-limiting step in the pathological effects of apoE4 on CA1 neurons is the accumulation of intracellular oligomerized Aβ42 which is mediated via a gain of function property of apoE4.

Following activation of the amyloid cascade, apolipoprotein E4 drives the in vivo oligomerization of amyloid-β resulting in neurodegeneration.

According to the amyloid hypothesis, the accumulation of oligomerized amyloid-β (Aβ) is a primary event in the pathogenesis of Alzheimers disease (AD). The trigger of the amyloid cascade and of Aβ oligomerization in sporadic AD, the most prevalent form of the disease, remains elusive. Here, we examined the hypothesis that apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for AD, triggers the accumulation of intraneuronal oligomerized Aβ following activation of the amyloid cascade. We investigated the intracellular organelles that are targeted by these processes and govern their pathological consequences. This revealed that activation of the amyloid cascade in vivo by inhibition of the Aβ degrading enzyme neprilysin specifically results in accumulation of Aβ and oligomerized Aβ and of ApoE4 in the CA1 neurons of ApoE4 mice. This was accompanied by lysosomal and mitochondrial pathology and the co-localization of Aβ, oligomerized Aβ, and ApoE4 with enlarged lysosomes and of Aβ and oligomerized Aβ with mitochondria. The time course of the lysosomal effects paralleled that of the loss of CA1 neurons, whereas the mitochondrial effects reached an earlier plateau. These findings suggest that ApoE4 potentiates the pathological effects of Aβ and the amyloid cascade by triggering the oligomerization of Aβ, which in turn, impairs intraneuronal mitochondria and lysosomes and drives neurodegeneration.

Pathological Synergism Between Amyloid-β and Apolipoprotein E4 – The Most Prevalent Yet Understudied Genetic Risk Factor for Alzheimer's Disease

This review focuses on apolipoprotein E4 (apoE4), the most prevalent genetic risk factor of Alzheimers disease, and on in vivo and in vitro model studies of the mechanisms underlying its pathological phenotype. The review will first center on in vivo studies with transgenic mice that express human apoE4 and other human apoE alleles, and on the extent to which this model mimics and reproduces the human apoE4 phenotypes. The second part of this review will address apoE4-related in vitro studies, with particular emphasis on the effects of the state of lipidation of apoE4 on its biochemical properties and on the extent to which the in vitro results can be generalized and applied to the in vivo situation. The third part of this review will focus on a novel pharmacological in vivo system that was recently developed in our laboratory, which is based on activation of the amyloid cascade in apoE transgenic mice by prolonged inhibition of the Abeta-degrading enzyme neprilysin and on what this system and its high spatio-temporal resolution has taught us about the mechanisms underlying the pathological effects of apoE4 in vivo.

Possible Role of Tau in Mediating Pathological Effects of apoE4 in vivo prior to and following Activation of the Amyloid Cascade

Injection of the neprilysin inhibitor thiorphan into the brain induces the accumulation of Aβ in hippocampal CA1 neurons and septal neurons in apoE4 knock-in mice but not in mice that express the corresponding Alzheimer’s disease benign isoform apoE3. We investigated the possible role of tau phosphorylation in mediating this synergistic pathological cross talk between apoE4 and the amyloid cascade. This revealed that in both apoE4 and apoE3 mice, activating the amyloid cascade by inhibiting neprilysin triggers the accumulation of AT100 phosphorylated tau in the perikarya of CA1 neurons. In contrast, in the septum this treatment elevated the level of phosphorylation of the tau AT100 epitope only in the apoE4 mice. This suggests that tau-related processes by themselves do not mediate the synergistic pathological effects of apoE4 and Aβ in CA1 neurons. However, tau and cytoskeletal-related mechanisms may mediate the synergistic pathological effects of apoE4 and Aβ in the septum. The basal levels of tau phosphorylation are also affected by the apoE genotype. This effect, which is associated with hyperphosphorylation of the tau AT8 epitope, is most prominent in hippocampal CA3 neurons. This suggests that the apoE4 mice are already stressed under nonstimulated conditions and that AT8 tau phosphorylation may contribute to their increased susceptibility to brain insults.

Activation of the Amyloid Cascade by Intracerebroventricular Injection of the Protease Inhibitor Phosphoramidon

We presently investigated the pathological effects of prolonged inhibition of brain β-amyloid (Aβ) degradation in vivo. The results obtained revealed that intracerebroventricular injection of the protease inhibitor phosphoramidon into wild-type mice for up to a month elevated the soluble and deposited brain Aβ levels and concomitantly induced the neurodegeneration of distinct hippocampal neurons as well as neuroinflammation. These findings reproduce pathological effects associated with the initial stages of the amyloid cascade and provide a novel model system for studying their underlying mechanisms.

Neuron-specific susceptibility to apolipoprotein E4.

The article by Schonheit et al. in this issue (2006) describes he effects of apoE4 on dendritic shape and geometry in he human hippocampus and discusses whether these effects re due to developmental differences in dendritic tree comlexity, or they appear in patients with Alzheimer’s disease AD) because of diminished capacity to withstand ageand D-related pathological changes. To minimize the possiility of secondary effects of AD pathology being exerted n the neuronal dendrites, the authors chose to focus on arvalbumin-positive GABAergic hippocampal interneurons f non-demented subjects with minimal AD pathology. Their tudy showed that, relative to apoE3, neither apoE4 nor apoE2 ignificantly affects dendritic morphology. This suggests that he dendritic tree of GABAergic hippocampal neurons is ot developmentally affected nor is it affected at a young ge by apoE4. The differentiation between clinically normal ubjects with mild AD pathology and normal aging is not learcut; the presently studied cases may thus contain both reclinical AD and normal aging subjects. However, previus studies revealed that the degeneration of parvalbuminontaining GABAergic interneurons in AD occurs late in the isease and is subsequent to the loss of other neuronal sysems [32], whereas other reports showed no effect on such eurons in AD [8,28]. These findings and results presented y Schonheit et al. in their present article both suggest that the arvalbumin-containing GABAergic neurons are relatively esistant to the pathological effects of AD and apoE4. The length and arborization of neuronal dendrites and heir plastic remodeling in AD are more severely affected in poE4 AD patients than in those who lack apoE4. The magitude of this effect is brain-area specific. For example, in

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deactivation pattern of MCI subjects was heterogeneous varying both according to their level of impairment and APOE-4 status. AD patients showed significantly less deactivation than OC in medial parietal and cingulate/retrosplenial cortices (z 5.32, p 0.0001). Comparing OC and AD subgroups, we found evidence of a hierarchy of deactivation decreasing in the following order: OC APOE-4 non-carriers OC APOE-4 carriers AD APOE-4 non-carriers AD APOE-4 carriers. Conclusions: The present study demonstrates that the pattern of task-related fMRI deactivation is remarkably disrupted in AD patients, particularly in AD APOE-4 carriers. Furthermore, consistent with PET findings, APOE-4 was associated with impaired parietal and posterior cingulate deactivation even in cognitively intact older individuals at risk for AD.