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Cell Culture System For Studying the Role of ApoE in Alzheimers Disease
Anna
G. Barsukova, Kenneth F. Gerhardt, Elin M. Grissom and Britto P. Nathan
Department
of Biological Sciences, Eastern Illinois University
Introduction
Apolipoprotein
(apo-)
E, a 34,000 molecular weight protein, exists in three major isoforms (apoE2,
apoE3, and apoE4) that are produced by three alleles (e2, e3, and e4) at a
single gene locus on chromosome 19. Through its function in transporting lipids
among cells, apoE plays a critical role in lipid metabolism within the body,
including the CNS. Recent findings demonstrate that inheritance of apoE4 allele
increases the risk of Alzheimers disease (AD). As a first step towards
understanding the role of apoE in the brain, we examined the effects of purified
human apoE3 and apoE4 on the growth of cultured adult mouse cortical (AMC)
neurons. We found major differences between apoE3 and apoE4 in regulating the
growth of neurites. Neurons grown in the presence of apoE3 had enhanced neurite
outgrowth, whereas neurons grown in the presence of apoE4 had stunted outgrowth.
Further studies revealed that there are significant differences between apoE3
and apoE4 in the amount and cellular localization in AMC neurons. What cell
surface protein regulates internalization of apoE3 and apoE4? Our studies
revealed that LRP is the primary receptor that mediates the internalization of
apoE in neurons.
Results
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Figure 1.
Quantification of human apoE3 (3 mg/ml) and apoE4 (3 mg/ml) in AMC
neurons at 37ēC and at 60 minutes at 4ēC. |
Cultured AMC neurons of two months
old male mice were treated with anti-human apoE primary antibody and secondary
fluorescent antibody to detect and quantify the amount of accumulated apoE
isoforms (n=3). Significant difference in intensity of optical density between
apoE3 and apoE4 was observed at all time intervals at 37ēC (p<0.05). No
significant difference was detected between apoE3 and apoE4 accumulation at 60
minutes period at 4ēC (p<0.05). The data are presented as the mean +/-
SEM. Repeated Measures ANOVA.
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Figure 2.
Distribution of apoE3 in AMC neuronal somata and dendrites at 37ēC and for 60
minute period at 4ēC. |
Significant difference detected for 15, 30, 60 minutes
intervals at 37ēC and at 60 minutes at 4ēC (p<0.05).The data are presented
as the mean ą SEM. Repeated Measures ANOVA.
 |
Figure 3.
Distribution of apoE4 in AMC neuronal somata and dendrites at 37ēC and at 60
minute period at 4ēC. |
In contrast to apoE3, apoE4 displayed significantly less
accumulation both in the cell bodies and neuritis (p<0.05). Most of the apoE4
was localized near the somata. Significant difference was detected for all time
courses between apoE4 accumulation in somata vs. dendrites (p<0.05). The data
are presented as the mean +/- SEM. Repeated Measures
ANOVA.
 |
Figure 4. Cell
surface binding and cellular accumulation of human apoE3 (3 mg/ml) and
apoE4 (3 mg/ml) in AMC neurons. Fluorescent microscopy, 100X. |
 |
Figure 5.
Effect of inhibitors of LRP receptor complex on accumulation of apoE3 and apoE4
in apoE KO AMC neurons at 60 minute time course at 37ēC. Fluorescent
microscopy, 100X. |
 |
Figure 6.
Quantification of apoE3 and apoE4 in apoE KO AMC neurons at 60 minute time
course, following the pretreatment of neurons with HSPG-LRP pathway inhibitors.
Inhibitors: RAP (5 mg/ml), lactoferrin (10 mg/ml), HSPG inhibitor heparinase I
(10 units/ml) at 37ēC, (n=3). |
Significant difference in apoE3 and apoE4
accumulation detected for RAP and lactoferrin treatments indicating that LRP
pathway is the major site of the differential binding of apoE isoforms
(p<0.05).The data are presented as the mean +/- SEM,
MANOVA.
Conclusions
ApoE3
accumulates more than apoE4 in AMC neurons
ApoE4
accumulates significantly less in dendrites than apoE3
Both apoE3
and apoE4 enters the neuron via the LRP |
