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Thesis
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Apolipoprotein E receptor and cholesterol level alterations are pronounced in Alzheimer’s Disease cortical synapses

UCLA Electronic Theses and Dissertations (2016)

Studies have identified amyloid beta (A?) protein accumulation in the synapse as a neurotoxic hallmark associated with synaptic dysfunction, which occurs early in Alzheimer’s Disease (AD) pathology. Clearance of soluble A? from the synapse is thought to occur through binding of A? to high density lipoprotein (HDL) and apolipoprotein E (apoE) complex in the brain. Studies suggest that the resulting A?/apoE/HDL cholesterol complex is a mechanism of internalizing and clearing A? from the synapse through apoE receptors, low density lipoprotein receptor (LDLR) and low density lipoprotein receptor related protein 1 (LRP1). This clearance process is thought to be dysfunctional in AD, however, evidence regarding specific molecular processes in the synaptic compartment involving apoE, A?, LDLR, LRP1, and cholesterol across AD stage and APOE genotype remains unclear. Therefore, levels of LDLR, LRP1, and cholesterol were measured in synaptosomes across AD stage and APOE genotype using flow cytometry and Western blot analysis.

Experimental results demonstrate that A? and apoE levels were increased in synaptosomes that were postitive for LDLR and LRP1 in late AD cases, which demonstrates possible co-localization of apoE receptors with A? and apoE. Additionally, LDLR was increased while LRP1 was reduced in late AD, suggesting that downregulation of LRP1 may contribute to dysfunction in A? clearance while increases in LDLR in late AD may be associated with a compensatory clearance mechanism for processes that is inefficient. Furthermore, esterified cholesterol was reduced while free cholesterol was increased in late AD versus normals, suggesting that increased lipid storage may play a role in AD prevention and free cholesterol in late AD may be associated with membrane instability and subsequent synaptic loss. APOE3/3 synaptosomes showed increased free cholesterol level and reduced esterified cholesterol level in late AD versus normals. APOE+e4 synaptosomes, however, showed no signficant cholesterol level changes across AD disease stage, implying that dynamic cholesterol turnover is necessary for synaptic health. Overall, the results suggest that apoE receptors and cholesterol level alterations are associated with AD pathogenesis via disruption of A? clearance mechanisms.

Cover page: Apolipoprotein E receptor and cholesterol level alterations are pronounced in Alzheimer’s Disease cortical synapses

Article
Peer Reviewed

Tau Spread, Apolipoprotein E, Inflammation, and More Rapidly Evolving Basic Science in Alzheimer Disease

UC San Francisco Previously Published Works (2017)

To date, Alzheimer disease drug candidates have produced negative results in human trials, and progress in moving new targets out of the laboratory and into trials has been slow. However, based on 3 decades of previous work, there is reason to hope that amyloid-based and other novel therapies will move at a faster pace toward successful clinical trials. This article highlights selected preclinical research topics that are rapidly advancing in the laboratory.

Cover page: Tau Spread, Apolipoprotein E, Inflammation, and More Rapidly Evolving Basic Science in Alzheimer Disease

Article
Peer Reviewed

Pre‐synaptic C‐terminal truncated tau is released from cortical synapses in Alzheimer's disease

UCLA Previously Published Works (2015)

The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from pre-synaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the pre-synaptic compartment in AD. Results demonstrate the abundance of tau, mainly C-terminal truncated tau, in synaptic terminals in aged control and in Alzheimer's disease (AD) samples. Tau fragments and dimers/oligomers are prominent in AD synapses. Following depolarization, tau release is potentiated in AD nerve terminals compared to aged controls. We hypothesize (i) endosomal release of the different tau peptides from AD synapses, and (ii) together with phosphorylation, fragmentation of synaptic tau exacerbates tau aggregation, synaptic dysfunction, and the spread of tau pathology in AD. Aβ = amyloid-beta.

Cover page: Pre‐synaptic C‐terminal truncated tau is released from cortical synapses in Alzheimer's disease

Article
Peer Reviewed

Apolipoprotein E/Amyloid-β Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4.

UC Irvine Previously Published Works (2019)

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.

Cover page: Apolipoprotein E/Amyloid-β Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4.

Creative Commons 'BY' version 4.0 license

Article
Peer Reviewed

Apolipoprotein E/Amyloid-β Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4

UC San Francisco Previously Published Works (2019)

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.