In the present study, we defined a physiological function of ApoEr2 at synapses. We demonstrated that ApoEr2 is expressed postsynaptically, and that ApoEr2 recruits and colocalizes with presynaptic specializations on contacting neuronal processes in COS7 cells. Moreover, ApoEr2 significantly increases dendritic spine density in primary hippocampal neurons, suggesting that ApoEr2 also contributes to spine development. We also examined how interaction between ApoEr2 and the synaptic adaptor proteins X11α and PSD-95 modulated ApoEr2-induced effects on synapse and dendritic spine formation. We found that X11α decreased, while PSD-95 increased, cell surface ApoEr2 levels. Additionally, we found that X11α inhibited, while PSD-95 enhanced ApoEr2-induced effects on synapses and spines. These results demonstrate that ApoEr2 is important for dendritic spine formation, and that this effect can be further modulated via interaction with its cytoplasmic adaptor proteins.
ApoEr2 plays an important role in induction of LTP, learning and memory, and synaptic transmission in adult brain 
. These processes require the cytoplasmic, alternatively spliced exon 19 of ApoEr2 
. In this study, we demonstrated the ability of ApoEr2 to recruit and colocalize with presynaptic specializations on contacting neuronal dendrites (), suggesting that ApoEr2 may be involved in synapse formation. This recruitment is often due to a trans-synaptic interaction involving either a homophilic or heterophilic adhesion 
. Since we observed that ApoEr2 induced synapse formation, we hypothesize that presynaptic Amyloid Precursor Protein (APP), which is expressed pre-and postsynaptically and known to associate with ApoEr2 
, or other presynaptic proteins may form trans-synaptic interactions with postsynaptic ApoEr2 to regulate synapse formation. There may also be extracellular molecules, such as F-spondin 
or Reelin 
, that affect the trans-synaptic interaction between ApoEr2 and pre-synaptic proteins to further regulate synapse formation. Additionally, it is possible that intracellular molecules, X11α and PSD-95, may alter ApoEr2's effects on synapse formation, as the present study demonstrates. However, further systematic analysis is necessary to determine the exact mechanism by which ApoEr2 acts.
We observed that ApoEr2 promotes dendritic spine formation in primary hippocampal neurons. To examine the effects of ApoEr2 on dendritic spine formation in vivo
, we conducted a Golgi rapid impregnation staining in ApoEr2 knockout mice and wild-type littermates at 1 month and 1 year of age. We found that 1 month old ApoEr2 knockout mice had significant deficits in spine number compared to controls (). However, 1 year old ApoEr2 knockout mice had similar spine densities compared to controls, suggesting that there may be compensatory mechanisms following initial spine formation in ApoEr2 knockout mice. One possible compensatory mechanism is modulation of extracellular ligands for ApoEr2, such as apolipoprotein E (apoE) or Reelin, which are known to be important for dendritic spine formation 
. Perhaps apoE and Reelin levels increased to compensate for ApoEr2 deficiency in ApoEr2 knockout mice, resulting in the similar spine densities compared to wild-type littermates that we observed at 1 year. It is also possible that ApoEr2 plays an important role in the initial formation of dendritic spines, but not their maintenance in adulthood. These possibilities can be further examined in the study of this receptor and its ligands as a function of age.
In the present study, we examined how ApoEr2 affects synapse formation by measuring synaptic protein levels of synaptophysin and PSD-95 in ApoEr2-infected neurons compared to controls. We observed that ApoEr2 infected neurons increased levels of PSD-95 and increased puncta of synaptophysin. Additionally, ApoEr2 overexpression increased protein levels of SPAR, a Rap-specific GAP that promotes spine formation (data not shown) 
. These data suggest that ApoEr2 may alter the number of synaptic sites in cultured hippocampal neurons by regulating the assembly of a complex of postsynaptic proteins. We also examined the effects of ApoEr2 on total and cell surface levels of AMPA receptor subunits and observed that ApoEr2 caused an increase in total and cell-surface levels of GluA2, but a decrease in total and cell surface levels of GluA1. These results are somewhat surprising given that GluA1/A2 heteromers comprise the predominant AMPA receptor population in the hippocampus 
. Our findings suggest that ApoEr2 may alter the subunit composition of AMPA receptors from a GluA1/A2 to a GluA2/A3 combination. While neither GluA1 nor GluA2 is absolutely required for normal dendritic spine formation or morphogenesis in vivo 
, GluA2 overexpression has been shown to enhance dendritic spine formation in hippocampal cultured neurons 
. Thus, the preferential increase in GluA2 by ApoEr2 could reflect a mechanism to stimulate spinogenesis during development.
Alternatively, a shift from GluA1 to GluA2-containing receptors could be related to differential cell biological properties of the two receptor subunits. For example, GluA1 has been suggested to require activity-dependent stimulation for acute delivery, whereas GluA2 may undergo constitutive trafficking and turnover 
. In addition, GluA1 channel conductance or opening is enhanced by phosphorylation during various forms of synaptic plasticity such as LTP 
. Furthermore, the great majority of GluA2 exists in a calcium impermeable isoform, such that regulation of GluA2 levels can serve as a mechanism to limit calcium permeability of AMPA receptors 
. Taken together, these divergent properties suggest that decreased GluA1 may lead to a reduced ability to undergo activity-dependent synaptic potentiation, perhaps as a compensatory response to the increase in number of excitatory synapses and dendritic spines generated by elevated GluA2. A better understanding of the functional consequences of AMPA receptor subunit composition shifts observed here will require additional studies conducting detailed electrophysiological analysis of ApoEr2-expressing neurons.
The definitive mechanism by which ApoEr2 acts remains unknown. It is possible that ApoEr2 modulates dendritic spine morphology by promotion of de novo spine formation or by stabilization of existing dendritic spines. We hypothesize that ApoEr2 may regulate spine formation through interaction with Reelin 
as well as through interaction with cytoplasmic adaptor proteins X11α and PSD-95. Supporting this hypothesis, we observed that the ligand binding domain of ApoEr2 is essential, but not sufficient for spine formation, suggesting that extracellular interaction with Reelin may be part of the mechanism by which ApoEr2 increases dendritic spine number. Furthermore, we found that co-transfection with ApoEr2 and PSD-95 enhanced dendritic spine formation as compared to ApoEr2 alone, as opposed to co-transfection with ApoEr2 and X11α, which decreased spine formation as compared to ApoEr2 alone. However, further investigation is necessary to determine how ApoEr2 affects synapses and spine formation.
In conclusion, our results demonstrate, for the first time, that ApoEr2 promotes presynaptic differentiation and dendritic spine formation in vitro and in vivo, an effect further regulated by interaction with cytoplasmic adaptor proteins. These findings provide a better understanding of the physiological actions of ApoEr2 in the normal brain. We speculate that these molecular roles may be relevant to ApoEr2 functions in synaptic plasticity and learning and memory, both of which depend on the dynamic or persistent formation or elimination of synapses and dendritic spines.