The high molecular weight immunophilin FKBP52 belongs to a family of versatile multi-domain proteins that are abundantly expressed in the nervous system and often show increased expression in damaged or degenerating brain regions. In the present study, we have identified a novel function of FKBP52 in Aβ-mediated toxicity using a genetic model in Drosophila
that expresses Aβ42 peptides. The protective effect of FKBP52 on Aβ toxicity during Drosophila
aging was evident from the observations that FKBP52
loss of function mutations potentiated Aβ toxicity, while over-expression of FKBP52
delayed or suppressed Aβ-induced phenotypes. Moreover, through genetic interactions and chemical approaches, we also found evidence that the effects of FKBP52 may be modulated by changes in intracellular copper homeostasis. These observations are consistent with our previous reports that FKBP52 interacts with the copper efflux machinery, and as such, establish a new aspect of involvement of the immunophilin family in Alzheimer's-related mechanisms. Several lines of evidence link the protective effects of FKBP52 with intracellular copper homeostasis. First, FKBP52 directly interacts with the copper metallochaperone Atox1 
, a protein that delivers copper to the copper transporting ATPAses ATP7A and ATP7B [reviewed in 35]
. Second, through genetic screens in this study, we found that mutations in the copper transport genes Ctr1A
, which directly regulate intracellular copper levels, modify Aβ-induced phenotypes in Drosophila
. Third, chemical manipulation of dietary copper levels also decreases or increases, respectively, the protective effect of FKBP52 on Aβ toxicity. Finally, MEF cells isolated from FKBP52
(−/−) mice show increased levels of copper compared to wild type MEF cells and over-expression of FKBP52 causes efflux of copper 
The metallobiology of copper plays a significant role in several neurodegenerative conditions. Interestingly, copper influences the aggregation properties of “toxic peptides” that contribute to these conditions, including beta amyloid, prion protein and α-synuclein as all three of these agents can bind copper ions [reviewed in 36]
. The presence of copper in mildly acidic conditions induces aggregation of the Aβ peptide 
and may exacerbate pathology linked to Aβ deposition. Our experiments show that all genetic manipulations that increased levels of copper, also enhanced the Aβ phenotypes. Copper manipulations were mediated either by increased copper entry, through over-expression of the plasma membrane transporter Ctr1A
, or by misregulation of cytoplasmic copper trafficking through loss-of-function mutations of the Atox1
genes. The enhancement of Aβ phenotypes by increasing dietary copper also supports these observations. Since copper is delivered to the trans-Golgi network (TGN) by the cytoplasmic transporter Atox1, we hypothesize that the interaction of Aβ with copper may at least partially take place in the TGN. Given the mildly acidic pH of the TGN 
, increased levels of copper in this compartment would lead to enhancement of Aβ toxicity and result in more severe Aβ-induced phenotypes, possibly through the induction of oxidative stress. Supporting the role of oxidative damage, over-expression of the anti-oxidative stress gene ferritin heavy chain suppressed the Aβ42-induced short lifespan in Drosophila 
. We further found that flies over-expressing dFKBP59
had lower levels of Aβ peptides, consistent with their suppressed phenotypes. Based on this, we suggest that dFKBP59 over-expression leads to increased Aβ turnover.
The interaction of FKBP52 with the transporter Atox1 presents a novel aspect of copper metabolism. FKBP52 participates in many cellular processes, including the translocation of steroid receptor complexes to the nucleus through interactions with dynein [reviewed in 40]
. FKBP52 also has chaperone activity shown by suppression of the aggregation of heat-denatured citrate synthase 
. We propose that FKBP52 may be required for the proper function of Atox1. Further analysis using double mutants of Atox1
and examining their effects on Aβ toxicity would be needed in order to confirm this hypothesis.
We also examined the effects of FKBP52 in mammalian cells expressing APP. Unlike Aβ, which does not contain proline amino acids and is not regulated by prolyl isomerization, the APP holo-enzyme binds the prolyl-isomerase Pin1 in its intracellular tail 
and it also interacts with the small immunophilin FKBP12 
. In the current studies we provide evidence that APP also binds FKBP52, via its FK506 binding domain and that FKBP52
(−/−) cells have higher levels of Aβ peptides than the same knock-out cells reconstituted with FKBP52
. The physical interaction of FKBP52 with APP suggests that this large immunophilin, in addition to altering Aβ levels, may have a role on the metabolism of APP. The effects of smaller members of the immunophilin family on the processing of APP would support such a role and future experimentation will address this hypothesis. In support of this novel role of immunophilins, we showed recently that a mutation in the Drosophila Ryanodine receptor homolog Rya-r44F
could modify a APP-overexpression associated phenotype 
. The FKBP12 protein interacts with ryanodine receptors 
, further implicating signaling through the immunophilin famlily with APP metabolism.
In summary, our studies show that the large immunophilin FKBP52 modulates Aβ toxicity, possibly through a mechanism that involves homeostasis of cellular copper. Our data does not rule out the possibility that the effects of FKBP52 mutations and metal transport act in parallel pathways, however, it provides indirect evidence for a possible mechanistic link between these respective pathways. Examination of effects of Aβ in FKBP52 knock-out mice will further validate our observations. The function of immunophilins is modulated by the FK506 family of ligands, several members of which have been developed to bind their targets without causing immune suppression. It will be interesting to examine if such ligands can modify the interaction of FKBP52 with APP and Aβ. We have evidence that the FK506-binding domain of FKBP52 is involved in the binding with APP, suggesting that immunophilin ligands may interfere with this interaction. Further studies will show whether these ligands are also involved in modulating toxicity of Aβ and may open the field for the development of a novel class of agents against Alzheimer's disease.