Numerous studies indicate that mutations in the presenilin genes play an important role in the development of early-onset FAD. PS1 and PS2 are members of a highly conserved family of TM proteins with sequence similarity to the C. elegans
Sel-12 and Spe-4 gene products. Recent studies suggest that PS1/2 proteins have six or eight TM domains with the N and C termini, as well as the largest hydrophilic loop region, facing the cytoplasmic compartment. Identification of a large number of mutations in the hydrophilic loop region suggests that this region is critical for presenilin function. We therefore set out to identify proteins that interact with the loop region of the presenilin proteins. Among the proteins identified that interact with both PS1 and PS2 are actin-binding protein ABP280 and a closely related protein, Fh1. The protein–protein interactions between the ABP280/Fh1 and PS1/2 are specific, because there were no detectable interactions observed between ABP280/Fh1 and LexA fusion proteins containing the intracellular domain of APP () or other unrelated bait proteins rich in hydrophilic amino acid residues (data not shown). The interactions between ABP280/Fh1 and PS1/2 proteins not only were detected in the yeast two-hybrid assay but also were confirmed in coimmunoprecipitation experiments. Interaction between PS1 and ABP280/Fh1 is supported further by the observation that overexpression of PS1 in Cos-1 cells by transient transfection leads to a change in the intracellular distribution of the endogenous ABP280/Fh1 proteins such that they colocalize with PS1. These results implicate a potential role of presenilin proteins in modulating the cytoskeleton in cells. Consistent with our results, it has been reported that the C-terminal fragments of both PS1 and PS2 are associated with cytoskeleton (Kim et al., 1997
Our results demonstrating interactions between presenilins and cytoskeletal-associated proteins ABP280/Fh1 provide insights into the possible functions of the presenilin proteins. ABP280 protein has been found to promote branching of actin filaments (Hartwig et al., 1980
; Niederman et al., 1983
; Hartwig and Shevlin, 1986
) and is proposed to modulate cell shape, polarity, and motility via changes in actin filament organization (Stossel, 1993
; Matsudaira, 1994
; Drubin and Nelson, 1996
; Mitchison and Cramer, 1996
). In eukaryotic cells the cytoskeleton is composed mainly of microtubules, microfilaments, and intermediate filaments. Biochemical and structural studies indicate that these polymeric systems are interacting with each other in cells (Pollard et al., 1984
; Green et al., 1987
). Actin-binding proteins such as ABP280 have been proposed to interconnect the microfilament and intermediate filament cytoskeletal systems (Brown and Binder, 1992
). Recently, the actin cytoskeleton has been implicated in regulating the activity of the cystic fibrosis TM conductance regulator (Prat et al., 1995
). It is conceivable that, by interacting with these cytoskeletal proteins, presenilins may modulate the activities of these and other related proteins in cells.
Both ABP280 and Fh1 are expressed in human brain tissue, as detected by Northern blotting and immunostaining. Our results of immunohistochemical staining of human brain sections indicate that both PS1 and ABP280/Fh1 proteins colocalize to astrocytes and delicate cellular processes in both gray matter and white matter of the cerebral cortex and hippocampal formation. Although we could not detect strong PS1 immunostaining in somatodendritic regions of neurons by using two different polyclonal antisera against PS1 protein on either paraffin or cryostat sections, strong PS1 immunoreactivity with both PS1 antisera was detected in fibrous astrocytes, consistent with a previous report that both mRNA and protein of PS1 are present in astrocytes (Lee et al., 1996
). In the AD brains there are more PS1-positive astrocytes distributed throughout the cortical laminae, as compared with the control brains. Immunostaining of astrocytes and fine cellular processes by the monoclonal antibody recognizing ABP280/Fh1 is similar to that detected by PS1 antibodies. Many studies suggest that astrocytes play an important role in the amyloid β
deposition and neurodegeneration observed in AD (Potter, 1992
; Nieto-Sampedro and Mora, 1994
; Pike et al., 1995
). Our study demonstrating that both PS1 and its interacting proteins ABP280/Fh1 are expressed in astrocytes raises the possibility that presenilin protein in astrocytes plays an important role in AD pathogenesis, providing additional support for the involvement of astrocytes in development of AD.
Robust ABP280/Fh1 immunoreactivity also was seen in blood vessels, NFTs, neuropil threads, and some senile plaque neurites. The specificity of the monoclonal antibody NCL-FIL is demonstrated by the observation that this antibody recognizes one protein band of 250 kDa, which may represent both ABP280 and Fh1, and that it did not cross-react with other proteins in the cell, including presenilin proteins, other cytoskeletal proteins, or unrelated proteins (NovoCastra Laboratories data) (; data not shown). Our PS1 antibodies did not show NFT immunoreactivity, although PS1 staining in NFTs has been reported (Murphy et al., 1996
). This discrepancy may reflect differences in the specificity of different PS1 antisera. NFTs are one of the major neuropathological characteristics of the AD brain. The accumulation of NFTs is highly correlated with the loss of pyramidal cells and with dementia. Ultrastructural and biochemical studies of NFTs have revealed that the major constituent of the paired helical filaments is polymerized and hyperphosphorylated tau (Wischik et al., 1995
). Molecular and immunohistochemical experiments demonstrated that NFTs also contain a number of cytoskeletal proteins, including neurofilament proteins, vimentin, actin, ubiquitin, and MAP2 (Wischik et al., 1995
). Because actin has been detected in NFTs, it is not surprising that actin-binding proteins are also present in NFTs. Our result that PS1 and PS2 interact directly with components of NFTs, ABP280/Fh1 proteins, raises the possibility that the presenilins may play an important role in the formation of NFTs. These observations also suggest that protein–protein interactions between presenilins and ABP280/Fh1 may be functionally significant. It is noted that only a subset of NFTs was detected by the filamin monoclonal antibody. In particular, in the AD brains we have examined, only intraneuronal NFTs, but not extraneuronal NFTs, were labeled by the filamin antibody. The exact mechanism of this apparent epitope selectivity is at present uncertain and awaits further investigation by comparing results from dual labeling and differential antigen retrieval and by using competing antigens.
Human ABP280 has been mapped to the X chromosome (Xq28) (Gorlin et al., 1993
; Maetrini et al., 1993
). The new filamin-related gene Fh1, which was identified in our study, maps to human chromosome 3. It is interesting to note that a putative locus for late onset FAD recently has been mapped to chromosome 3, although detailed information is not available yet (Tanzi et al., 1996
). The protein–protein interactions between Fh1 and presenilin proteins and the presence of Fh1 in NFT in the AD brain suggest that Fh1 is a candidate FAD gene. We are presently testing whether Fh1 could be the putative AD locus on chromosome 3.