Fragmentation of Apolipoprotein E4 is Required for Differential Expression of Inflammation and Activation Related Genes in Microglia Cells

Abstract

The apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD) and accumulating evidence suggests that fragmentation with a toxic-gain of function may be a key molecular step associated with this risk. Recently, we demonstrated strong immunoreactivity of a 151 amino-terminal fragment of apoE4 (E4-fragment) within the nucleus of microglia in the human AD brain. In vitro, this fragment led to toxicity and activation of inflammatory processes in BV2 microglia cells. Additionally, a transcriptome analysis following exogenous treatment of BV2 microglia cells with this E4 fragment led to a > 2-fold up regulation of 1,608 genes, with many genes playing a role in inflammation and microglia activation. To extend these findings, we here report a similar transcriptome analysis in BV2 microglia cells following treatment with full-length ApoE4 (FL-ApoE4). The results indicated that full-length ApoE4 had a very small effect on gene expression compared to the fragment. Only 48 differentially expressed genes (DEGs) were identified (p < 0.05, and greater than 2-fold change). A gene ontology analysis of these DEGs indicated that they are not involved in inflammatory and activation processes, in contrast to the genes up regulated by the E4-fragment. In addition, genes that showed a negative fold-change upon FL-E4 treatment typically showed a strong positive fold-change upon treatment with the fragment (Pearson's r = -0.7). Taken together, these results support the hypothesis that a key step in the conversion of microglia to an activated phenotype is proteolytic cleavage of FL-ApoE4. Therefore, the neutralization of this amino-terminal fragment of ApoE4, specifically, may serve as an important therapeutic strategy in the treatment of AD.

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish

“…These results support our previous in vitro findings indicating enhanced cellular toxicity of only nApoE4 1-151 , which differs by a single amino acid at position 112 (R>C) [17]. The further identification of nApoE4 1-151, within the nucleus of neurons of the developing nervous system of zebrafish supports the hypothesis that the uptake of this fragment and trafficking to the nucleus may lead to stimulation of cell death pathways similar to what we have recently observed in BV2 microglia cells [15,21].…”

“…Although ApoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis remains speculative. Recent data from our lab suggests that, in vitro , nApoE4 1-151 can traffic to the nucleus leading to toxicity and expression of inflammatory genes in BV2 microglia cells [15, 17, 21]. In the present study, we now expand those findings in vivo , by demonstrating toxicity of nApoE4 1-151 , developmental abnormalities, and motor behavior deficits in a novel model system consisting of zebrafish.…”

“…Second, generation of this fragment was documented following incubation of full-length ApoE4 with matrix metalloproteinase-9 (MMP-9) [17]. Finally, Recent data from our lab suggests that, in vitro , a 151 amino-terminal fragment of ApoE4 (nApoE4 1-151 ) can traffic to the nucleus leading to toxicity and expression of inflammatory genes in BV2 microglia cells [15, 16, 21]. The purpose of this current study is to expand those findings in vivo , by assessing the mechanisms by which this fragment may induce toxicity, developmental abnormalities, and behavior deficits in a model system consisting of zebrafish.…”

“…Additional studies have also shown neurotoxic and pro-inflammatory responses to the fragmentation of ApoE4 [26-29]. Recently we identified a 151 amino-terminal fragment of ApoE4 (nApoE4 1-151 ) that localized within the nucleus of both neurons and microglia cells of the human AD brain [17] and in vitro , we demonstrated this fragment is taken up by BV2 microglia cells, traffics to the nucleus and leads to the upregulation of thousands of genes, many of which associated with microglia activation and inflammation [15, 21]. In the current studies, we expanded these findings utilizing an in vivo model system consisting of zebrafish.…”

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish

“…Second, generation of this fragment was documented following incubation of full-length ApoE4 with matrix metalloproteinase-9 (MMP-9) [ 18 ]. Finally, recent data from our lab suggests that, in vitro , nApoE4 1-151 can traffic to the nucleus leading to toxicity and expression of inflammatory genes in BV2 microglia cells [ 16 , 17 , 28 ]. It is noteworthy that because zebrafish embryos do not yet express fully functional glial cells at 24 hpf including microglia [ 29 ], our study focused on the effects of nApoE4 1-151 on neuronal populations.…”