The present study provides support for a pathologic role of the ABAD-Aβ interaction in promoting mitochondrial and neuronal dysfunction in an Aβ-rich environment, as shown in a mouse model of AD. We have demonstrated substantial evidence of the protective effect of blocking the ABAD-Aβ interaction using a combination of genetic and biochemical approaches applied to this mouse model.
Initially, using the yeast two-hybrid system, and, subsequently, by ligand binding assays with purified polypeptides and crystal structure analysis, our group identified an ABAD polypeptide capable of binding Aβ (Yan et al., 1997
; Lustbader et al., 2004
). Next, we identified an ABAD-Aβ complex in vivo
in cortical mitochondria from AD brain and transgenic AD mice (Lustbader et al., 2004
). Importantly, the ABAD-Aβ interaction exacerbates mitochondrial and neuronal dysfunction in AD mice and in Aβ-induced neuronal injury (Yan et al., 1999
; Lustbader et al., 2004
; Takuma et al., 2005
; Yan and Stern, 2005
), indicating that the interaction of ABAD with Aβ is critical for facilitating Aβ-induced mitochondrial and neuronal stress. These studies raised the question as to whether the blockade of the ABAD-Aβ interaction might attenuate these deleterious effects observed in APP/Aβ mice or in Aβ-perturbed neurons.
To block ABAD-Aβ interaction, we used an ABAD decoy peptide to compete with and neutralize the binding of Aβ to endogenous full-length ABAD. The choice of peptide was guided by structural studies demonstrating that residues (94–114) of ABAD comprise the site of Aβ binding, and a peptide encompassing this region (residues 92-120) of human ABAD (ABAD-DP) inhibited binding of ABAD to Aβ based on surface plasmon resonance (Lustbader et al., 2004
). Further in vitro
experiments revealed that the addition of cell-permeable ABAD-DP, the latter with the cell-membrane transduction domain of HIV Tat protein, to cultured cortical neurons largely prevented Aβ-induced cytochrome c
release, ROS production, and apoptosis (Lustbader et al., 2004
In the present study, we demonstrate the protective effects of inhibition of ABAD-Aβ binding on mitochondrial properties and cognitive function in an in vivo
setting. First, using a recombinant molecular technique, we constructed a biologically active ABAD(93-116) peptide fused to the HIV Tat transduction protein domain and a mitochondrial targeting sequence. The protein transduction domain embedded in the HIV Tat protein (47–57) has been successfully used to study intracellular mechanisms based on high efficiency (~90%) delivery of peptides/polypeptides both in vitro
and in vivo
(Lissy et al., 2000
; Aarts et al., 2002
; Cao et al., 2002
; Wadia and Dowdy, 2002
). It has been shown that Tat-linked proteins can penetrate the blood–brain barrier; thus Tat-mediated delivery of proteins/peptides holds potential for future therapeutic applications. The presence of the membrane transduction and mitochondrial targeting domains results in effective penetration and concentration of polypeptides into mitochondria in an intact tissue. As we observed, Tat-ABAD peptide was efficiently transduced to neurons and localized to mitochondria in vitro
and in vivo
Indeed, we have successfully used this decoy peptide before to demonstrate in vivo
effects. Specifically, from a proteomic study of proteins with elevated expression in Tg mAPP/ABAD mice, we identified two proteins, peroxiredoxin II and endophilin I, present at higher levels in the cerebral cortex of Tg mAPP, Tg mAPP/ABAD mice and human AD patients (Yao et al., 2007
; Ren et al., 2008
). These two proteins typify the complex changes in protein expression in AD brain, as the expression of peroxiredoxin II and endophilin I has the potential to either promote or inhibit neuronal survival, respectively (Yao et al., 2007
; Ren et al., 2008
). Using the expression of these two proteins as a biological marker, we showed that the Tat-Mito-ABAD-DP decoy peptide, when injected intraperitoneally into Tg mAPP mice (using the same protocol as in the current study) had the ability to diminish expression of these two proteins to normal levels while the reverse peptide was inactive in this regard (Yao et al., 2007
; Ren et al., 2008
In this study, we have gone further and directly observed that introduction of ABAD decoy peptide in vivo dissociates ABAD from Aβ with resultant restoration of ABAD function and PreP activity. In parallel, treatment with the ABAD decoy peptide decreased formation of the ABAD-Aβ complex, restored mitochondrial respiratory function and enzyme activity, attenuated mitochondrial ROS production/accumulation, increased activity of Aβ degrading enzyme, and, presumably, thereby improved learning and memory. Similar results were observed in Tg mAPP mice overexpressing neuronal ABAD decoy peptide. Together, these observations confirm the protective effects of ABAD decoy peptide.
Intriguingly, mitochondrial Aβ levels were significantly lower in Tg mAPP mice treated with the ABAD decoy peptide or in mice expressing this peptide versus vehicle-treated mAPP mice. Furthermore, PreP proteolytic activity was decreased in Aβ-rich mitochondria, but was significantly enhanced in the presence of the ABAD decoy peptide. It has been demonstrated that human PreP is the sole Aβ-degrading protease responsible for degradation and clearance of Aβ in mitochondria recognized to date. Impaired PreP activity would therefore promote accumulation of Aβ, ultimately causing aberrant mitochondrial and neuronal function (Falkevall et al., 2006
). In situ
immunoinactivation studies using anti-PreP antibody eliminated degradation of Aβ, proving that PreP is necessary for the clearance of Aβ in mitochondria. Thus, decreased PreP activity may be an important mechanism underlying Aβ accumulation in mitochondria in mAPP mice.
Given that proteolytic activity of PreP under oxidizing conditions was associated with Aβ accumulation (Falkevall et al., 2006
), we predicted that diminished PreP activity, observed in the presence of ABAD-Aβ complex (i.e., in Tg mAPP mice), would occur in tandem with higher levels of mitochondrial ROS. Consistent with this concept, in the presence of ABAD-DP (which dissociated the complex between full-length ABAD and Aβ), levels of ROS were reduced and PreP demonstrated the ability to degrade Aβ. These data are also consistent with the changes in mitochondrial Aβ levels in Tg mice, which were elevated in Tg mAPP mice and reduced by administration of mito-ABAD-DP.
Together, these findings provide substantial evidence of the protective effect of inhibition of ABAD-Aβ interaction on mitochondrial, neuronal, and cognitive function in an AD mouse model. Most importantly, we demonstrated that blockade of ABAD-Aβ interaction reduces mitochondrial Aβ accumulation, leading to improvement in mitochondrial function, attenuation of mitochondrial ROS production, and increased PreP activity. Elevated levels of oxidative stress in mitochondria due to a direct effect of Aβ or ABAD-Aβ interaction may be responsible for decreased PreP activity, which in turn leads to further accumulation of Aβ, and exacerbates mitochondrial and neuronal dysfunction, all of which contribute to the picture of AD pathogenesis. These results provide the first mechanistic insight into Aβ perturbation of mitochondrial function in AD. One or more agents that block ABAD-Aβ interaction may therefore be potential therapeutic approaches for preventing and/or halting AD progression.