Human APOE genotype affects intraneuronal Aβ1-42 accumulation in a lentiviral gene transfer model - PubMed (original) (raw)

. 2014 Mar 1;23(5):1365-75.

doi: 10.1093/hmg/ddt525. Epub 2013 Oct 23.

Affiliations

Human APOE genotype affects intraneuronal Aβ1-42 accumulation in a lentiviral gene transfer model

Wenjuan Zhao et al. Hum Mol Genet. 2014.

Abstract

Intraneuronal accumulation of β-amyloid (Aβ)42 is one of the earliest pathological events in humans and in animal models of Alzheimer's disease (AD). Apolipoprotein E 4 (APOE4) is the major identified genetic risk factor for late-onset AD, with Aβ deposition beginning earlier in apoE4-positive subjects. To directly determine the effects of APOE genotype on intraneuronal accumulation of Aβ1-42 at the onset of AD pathogenesis, we introduced lentiviral Aβ1-42 into the cortex of APOE targeted replacement (TR) mice at the age of 8-9 months. We demonstrated a significant isoform-dependent effect of human APOE, with dramatically enhanced intracellular Aβ1-42 deposits in the cerebral cortex of APOE4-TR mice 2 weeks after injection. Double-immunofluorescent staining showed that intracellular accumulation of lentiviral Aβ1-42 was mainly present in neurons, localized to late endosomes/lysosomes. This intraneuronal accumulation of Aβ1-42 correlated with increased tau phosphorylation and cell death in the ipsilateral cortex around the injection site. Aβ1-42 was also observed in microglia, but not in astrocytes. Quantitative analysis revealed more neurons with Aβ1-42 while less microglia with Aβ1-42 nearest to the injection site of Aβ1-42 lentivirus in APOE4-TR mice. Finally, apoE was present in neurons of the ipsilateral cortex of APOE-TR mice at 2 weeks after lentivirus injection, in addition to astrocytes and microglia in both the ipsilateral and contralateral cerebral cortex. Taken together, these results demonstrate that apoE4 tips the balance of the glial and neuronal Aβ toward the intraneuronal accumulation of Aβ.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Microinjection and intraneuronal expression of lentiviral Aβ1–42. (A) The schematic of the microinjection site of lentiviral Aβ1–42 and its distribution in the mouse cortex. The stereotaxic coordinates for the primary motor cortex of mouse were 1.6 mm lateral (left), 1.6 mm ventral and 0.5 mm anterior. Aβ1–42 viral stocks (6 μl) were injected through a microsyringe pump controller at a rate of 0.2 μl/min. (B and C) showed MOAB2 immunofluorescent staining in the contralateral (right) cortical area of a C57BL/6J mouse brain section without expression of lentiviral Aβ1–42 at magnifications of 20× and 40×. (D–F) showed Representative images of MOAB2 immunofluorescent staining in ipsilateral (left) cortex near the injection area of the mouse brain section at magnifications of 20×, 40× and 100×, respectively. In the higher magnifications, the staining of lentiviral Aβ1–42 was intracellular with a punctate pattern. Scale bars: B, D, 50 μm; C, E, 20 μm; F, 10 μm.

Figure 2.

Figure 2.

Intracellular accumulation of lentiviral Aβ1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. (A) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ1–42 from APOE4-TR mouse. Scale bar = 500 μm. (B) The percentage of MOAB2-positive sections was significantly higher in APOE4-TR mice than that in APOE2- or APOE3-TR mice (**P < 0.01, APOE4+/+ versus APOE2+/+, APOE4+/+ versus APOE3+/+, Fisher's exact test). (C) The mean integrated density of MOAB2-positive staining in the ipsilateral cortex of APOE4-TR mice was significantly higher than that of APOE2- or APOE3-TR mice. The intensity of DAB stained images at 10× magnification was quantified by using the Image J software. Data are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Tukey's post hoc analyses. **P < 0.01, ipsilateral cortex of APOE4+/+ versus ipsilateral cortex of APOE2+/+ and APOE3+/+, ##P < 0.01, ipsilateral cortex of APOE4+/+ versus contralateral cortex of APOE4+/+. (D) Combination of DAB-labeling immunohistochemistry for MOAB2 (brown) with Nissl staining (blue) in the ipsilateral cortex near the injection site of Aβ1–42 lentivirus on a brain section of APOE4-TR mouse. Arrows and arrowheads indicate MOAB2 immunopositive staining in neuron- and glia-like cells, respectively. (E) Combination of DAB staining for MOAB2 (brown) with Nissl staining (blue) in the contralateral cortex on a brain section of APOE4-TR mouse. (F) Representative images of DAB staining for phospho-tau pSer199/202 antibody (AT8, brown) with Nissl staining (blue) in the ipsilateral cortex near the injection site of Aβ1–42 lentivirus on a brain section of APOE4-TR mouse. Arrows indicate phosphorylated tau in neurons. Solid arrowheads indicate phosphorylated tau in the absence of Nissl staining. Blank arrowheads indicate phosphorylated tau in neuronal processes. (G) Combination of DAB staining for AT8 antibody (brown) with Nissl staining (blue) in the contralateral cortex on a brain section of APOE4-TR mouse. Scale bars = 50 μm.

Figure 3.

Figure 3.

Lentiviral Aβ1–42 is mainly present in neurons, to a lesser extent in microglia, but not in astrocytes at 2 weeks after injection of lentivirus into the brain cortex of APOE2-, APOE3- and APOE4-TR mice. (A) Aβ1–42 (MOAB2, red) mainly deposited in cortical neurons (NeuN, green), displayed a punctate perinuclear localization. (B) Aβ1–42 (MOAB2, red) was also present in some microglia (Iba1, green). (C) Aβ1–42 (MOAB2, red) was not observed in astrocytes (GFAP, green). Scale bar = 10 μm.

Figure 4.

Figure 4.

APOE genotype affects Aβ1–42 accumulation in neurons and glia. (A) Average number of MOAB2-positive neurons per mouse in the ipsilateral cortex near the injection site of Aβ1–42 lentivirus. (B) The average numbers of microglia per mouse were determined from Iba1 stains. (C) MOAB2-positive microglia (MOAB2-/Iba1-double-positive cells) in the ipsilateral cortex near the injection site of Aβ1–42 lentivirus were determined per mouse. Data were expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's post hoc analyses. *P < 0.05, **P < 0.01.

Figure 5.

Figure 5.

Intraneuronal accumulation of lentiviral Aβ1–42 is mainly localized to late endosomes/lysosomes, not early endosomes, in the ipsilateral cortex at 2 weeks after lentiviral injection. (A) Intraneuronal Aβ1–42 detected with MOAB2 (red) co-localized with late endosomal/lysosomal marker ABL-93 (anti-LAMP-2, green) in the ipsilateral cortex of an APOE4-TR mouse. (B) Intraneuronal Aβ1–42 (MOAB2, red) did not obviously co-localize with early endosomal marker EEA1 (green) in the ipsilateral cortex of an APOE4-TR mouse. Scale bar = 10 μm. Colocalization of antibodies appears as yellow.

Figure 6.

Figure 6.

Immunohistochemistry of apoE in the brain cortex of APOE-TR mice at 2 weeks after injection of lentiviral Aβ1–42. (A) ApoE-positive cells (green) in both the ipsilateral and contralateral brain cortex. (B) In the ipsilateral cerebral cortex, apoE (green) is present in Aβ1–42-positive neurons (MOAB2, red), as well as microglia (Iba1, red) and astrocytes (GFAP, red). In the contralateral cerebral cortex, apoE is mainly present in microglia and astrocytes. Scale bar = 20 μm.

Similar articles

Cited by

References

    1. LaFerla F.M., Green K.N., Oddo S. Intracellular amyloid-beta in Alzheimer's disease. Nat. Rev. Neurosci. 2007;8:499–509. - PubMed
    1. Bayer T.A., Wirths O. Intracellular accumulation of amyloid-beta—a predictor for synaptic dysfunction and neuron loss in Alzheimer's disease. Front. Aging Neurosci. 2010;2:8. - PMC - PubMed
    1. Gouras G.K., Tampellini D., Takahashi R.H., Capetillo-Zarate E. Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer's disease. Acta Neuropathol. 2010;119:523–541. - PMC - PubMed
    1. Mohamed A., Posse de Chaves E. Aβ internalization by neurons and glia. Int. J. Alzheimers Dis. 2011;2011:127984. - PMC - PubMed
    1. Gouras G.K., Tsai J., Naslund J., Vincent B., Edgar M., Checler F., Greenfield J.P., Haroutunian V., Buxbaum J.D., Xu H., Greengard P., Relkin N.R. Intraneuronal Abeta42 accumulation in human brain. Am. J. Pathol. 2000;156:15–20. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources