Proapoptotic Mitochondrial Carrier Homolog Protein PSAP Mediates Death Receptor 6 Induced Apoptosis

Abstract

Presenilin-associated protein (PSAP) was originally identified as a mitochondrial proapoptotic protein. To further explore the apoptotic pathway that involves PSAP, our yeast two-hybrid screen revealed that PSAP interacts with a death receptor, DR6. DR6 is a relatively less common member of the death receptor family and has been shown to mediate the neurotoxicity of amyloid-β, mutant SOD1, and prion proteins and has also been implicated in the regulation of immune cell proliferation and differentiation. Our previous study showed that DR6 induces apoptosis via a unique mitochondria-dependent pathway different from the conventional death receptor-mediated extrinsic apoptotic pathways. Thus, the interaction of DR6 with PSAP established a direct molecular link between DR6 and mitochondrial apoptotic pathway. We investigated the possible role of PSAP in DR6-induced apoptosis. Interestingly, it was discovered that knockdown of PSAP strongly inhibited DR6-induced apoptosis. To further elucidate the mechanism by which PSAP mediates DR6-induced mitochondria-dependent apoptosis, our data demonstrated that knockdown of PSAP blocked DR6-induced Bax translocation and cytochrome c release from the mitochondria. Moreover, it was found that both PSAP and DR6 form complexes with Bax, but at different subcellular locations. The DR6-Bax complex was detected in the cytosolic fraction while the PSAP-Bax complex was detected in the mitochondrial fraction. The observation that knockdown of DR6 significantly reduced the amount of PSAP-Bax complex detected in mitochondria suggests a possibility that DR6-bound Bax is transferred to PSAP upon interaction with PSAP at the mitochondria, leading to cytochrome c release and eventually apoptosis.

Keywords

Alzheimer’s disease amyotrophic lateral sclerosis apoptosis DR6 mitochondria neurodegenerative disease PSAP

INTRODUCTION

Most of the early-onset familial forms of Alzheimer’s disease (AD) have been linked to the mutations in the genes encoding presenilin 1 (PS1) [1] and presenilin 2 (PS2) [2]. Both PS1 and PS2 function as the catalytic subunit of the γ-secretase complex, which catalyzes the processing of amyloid-β protein precursor (AβPP), resulting in the formation of amyloid-β peptide (Aβ). Besides γ-secretase activity, PS1 and PS2 have also been implicated in apoptotic cell death [3]. To determine the mechanism by which PS1 is involved in apoptosis, our previous study identified a PS1 associated protein (PSAP) [4]. Sequence analysis revealed that PSAP contains six putative transmembrane domains and shares a high degree of sequence homology at the amino acid level with the consensus sequence of mitochondrial carrier proteins, a family of proteins that reside in the mitochondria and catalyzes the transport of metabolites across the inner mitochondrial membrane; thus, PSAP is now also known as mitochondrial carrier homolog 1 (MTCH1) [5, 6]. Subsequently, PSAP was found to localize in the mitochondrial outer membrane and causes apoptosis by induction of cytochrome c release when it is overexpressed [5, 6]. More interestingly, PSAP induces apoptosis in a Bax/Bak independent manner [7, 8], suggesting that overexpression of PSAP evokes apoptosis through a unique mitochondrial pathway that is not controlled by the Bcl-2 family protein [8]. To further determine the molecule(s) involved in PSAP-regulated apoptosis, by employing the yeast two-hybrid system, our current study revealed that PSAP interacts with death receptor 6 (DR6).

DR6, also known as TNFRSF21, is a relatively new member of the TNF receptor super family (TNFRSF) and induces apoptosis upon overexpression [9, 10]. It is noteworthy that recent studies suggested that DR6 regulated apoptotic pathway may contribute to the neurodegeneration observed in AD by mediating the neurotoxicity of Aβ and the N-terminal fragment of AβPP [11, 12]. DR6 was also implicated in prion protein-induced axonal degeneration [13], and mutant SOD1 (superoxide dismutase 1)-induced motor neuron cell death observed in amyotrophic lateral sclerosis (ALS) [14]. Moreover, recent studies showed that DR6 promotes Wallerian degeneration in peripheral axons [15] and negatively regulates the maturation and survival of oligodendrocytes, which are the myelinating cells of the central nervous system [16]. In addition to neuronal cells, DR6 has been shown to control proliferation and differentiation of immune cells and contribute to autoimmunity [17 –20]. Studies also suggest that the DR6 pathway plays a major role in the regulation of hepatocyte apoptosis [21] and contributes to tumor cell-induced endothelial cell necroptosis, promoting metastasis [22]. Despite the important roles DR6 plays in these pathological events, the molecular mechanisms by which DR6 induces apoptosis and/or necroptosis remain elusive. Our recent study strongly suggested that DR6 induces apoptosis exclusively through a unique mitochondrial pathway and depends on Bax translocation [9]. However, the intracellular molecule(s) that transduce the DR6-induced death signal and mediate DR6-induced Bax translocation remain unknown. In the current study, our data demonstrated that DR6 interacts with a novel proapoptotic mitochondrial protein PSAP. More interestingly, knockdown of PSAP strongly inhibited DR6-induced apoptosis. Our data also showed that both DR6 and PSAP could form complex with Bax, but at different subcellular locations and that knockdown of DR6 reduced the level of PSAP-Bax complex formation, suggesting that PSAP functions as an anchor to receive DR6-delivered Bax on the mitochondria. Thus, these findings established for the first time a molecular link between DR6 and the mitochondrial apoptotic pathway.

MATERIALS AND METHODS

Reagents

Lipofectamine 2000 transfection reagent and Mitochondria isolation kit were purchased from Thermo Fisher. Complete Protease Inhibitor Cocktail Tablets were purchased from Bimake. Anti-Myc, anti-β-actin, and anti-COX I antibodies were purchased from Santa Cruz Biotechnology. Antibodies against poly (ADPribose) polymerase (PARP), caspase-3, -8, -9, Bax, Bak, Bid, Bmf, Bim, Puma, and Smac were purchased from Cell Signaling Technology. Anti-DR6 antibody was purchased from R&D Systems. Anti-cytochrome c antibody was purchased from BD Biosciences. Both anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Bioss. Streptolysin O was purchased from Sigma. Recombinant human tumor necrosis factor α (TNFα) was purchased from Calbiochem (La Jolla, CA, USA). pcDNA3.1/LacZ-Myc and pcDNA3.1/DR6-Myc plasmids were amplified as described previously [9].

Yeast two-hybrid and cDNA library screening

Yeast two-hybrid and cDNA library screening was performed as described previously [4]. All yeast strains, plasmids, and the human brain cDNA library used were purchased from CLONTECH as components of the Matchmaker two-hybrid system. The full-length PSAP polypeptide was fused to the GAL4 DNA binding domain by subcloning the PSAP cDNA into the pAS2-1 vector (pASPSAP). Yeast strain Y190 was co-transformed with the plasmid pASPSAP and a human brain cDNA library fused to the GAL4 transcription activation domain in pACT2 vector. Two-hybrid screening was carried out according to the manufacturer’s instructions (CLONTECH).

Galactosidase assays

The following plasmids were used in yeast mating experiments. Positive control plasmids pVA3-1 (p53 gene in pAS2-1), pTD1-1 (SV40 large T antigen in pACT2), and negative control pLAM5’-1 (the lamin C gene in pAS2-1) from CLONTECH were used as controls. pASPSAP was used as a bait. Plasmids pAC(10-16) and pAC(11-9) in pACT2 vector were isolated from the two positive clones 10–16 and 11–9, respectively. Plasmids based on the pAS2-1 vector were used to transform the yeast strain Y187. Plasmids based on the pACT2 vector were used to transform the yeast strain Y190. The mating experiments and color development assays were performed according to the manufacturer’s instructions (CLONTECH).

Cell culture and transfection

HeLa cells were cultured at 37°C and 5% CO2 in DMEM (Hyclone) supplemented with 10% fetal bovine serum, and 100 units/ml penicillin, and 100 μg/ml streptomycin. Cell transfections were carried out using Lipofectamine 2000 following the manufacturer’s instructions. TRE-HeLa stable inducible cells were established as described previously [9].

siRNA treatment

siRNAs specific to Bax and PSAP as well as the control siRNA and siRNA reagent were purchased from GenePharma (Shanghai). T-RE HeLa inducible cells were transfected with these siRNAs for 48 h, following the instructions provided by the manufacturer. On the third day, cells were induced for DR6 expression. 24 h after induction, cells were harvested and lysed for further analysis.

Subcellular fractionation

Cytosolic and the mitochondria-containing fractions were prepared by permeabilization of cells with streptolysin O as described previously [9]. Mitochondrial faction was extracted using mitochondria isolation kit (from Thermo fisher) for mitochondria preparation.

Western blot and immunoprecipitation

Western blots and immunoprecipitations were performed as described previously [9].

Densitometry and statistics

Densitometric analysis was performed using Gel Digitizing Software UN-SCAN-IT (Silk Scientific, Orem, UT, USA) as described in our previously [23]. Data were expressed as means±standard error of the mean (SEM). Significant differences among groups were analyzed using a one-way ANOVA with PRISM statistical analysis software (GraphPad Software). Differences were considered statistically significant at p < 0.05.

RESULTS

Proapoptotic mitochondrial protein PSAP interacts with DR6 and Bax in TNFα-induced apoptosis

To determine the apoptosis pathway that involves PSAP, we performed a yeast two-hybrid system analysis. Using PSAP as a bait, a brain cDNA library of 3.1×10⁶ total clones was screened for PSAP interacting protein(s). As shown in Fig. 1, two of the three positive clones, 10–16 and 11–9 were isolated and further characterized. Sequence analysis revealed that clone 10–16 encodes the PS1 protein, which is known to interact with PSAP [4], while clone 11–9 was found to contain a 2.8 kb cDNA fragment that encodes the full-length TNFRSF21 (also known as DR6) of 655 amino acids. To further confirm the association of PSAP with DR6, we performed the co-immunoprecipitation experiments. HeLa cells were treated with TNFα, which is a ligand of the TNF receptors and has been shown to induce DR6 expression and provoke apoptosis [24], and the cell lysate was subjected to immunoprecipitation using antibodies against PSAP or DR6. As shown in Fig. 2A, DR6 was co-immunoprecipitated with PSAP by a PSAP-specific antibody (line 4), clearly indicating that PSAP forms complexes with DR6. In addition, when HeLa cells were treated with TNFα, besides DR6, the proapoptotic Bcl-2 family members Bax and Bak, which contains three BH domains, were also co-immunoprecipitated with PSAP by several PSAP-specific antibodies, but not the BH3-only proapoptotic members, such as tBid, Bmf, Bim, and Puma (Fig. 2B, C). Furthermore, as shown in Fig. 2D, upon treatment with TNFα, cytochrome c was released from mitochondria to the cytosol; Bax and tBid were translocated from cytosol to mitochondria. These data strongly suggest a possible role for PSAP in TNFα-induced and DR6-mediated apoptosis.

Fig.1

Identification of DR6 as a PSAP-interacting protein by yeast two-hybrid screening. To eliminate false positive, yeast-mating experiments were carried out. A) The transformant pair that bears the positive control plasmids as well as the transformant pair bears the bait plasmid pAPSAP and plasmid pAC(10–16) were positive for the His3 phenotype and for the β-galactosidase activity, indicating a true positive interaction between PSAP and the peptide expressed by pAC(10–6). As expected, the negative control pair bearing plasmids pLAM5’-1 and pAC(10–6) was unable to grow on plate lacking amino acid His in culture media. B) Similarly, the true positive interaction between PSAP and the protein encoded by clone 11–9 was confirmed by this yeast mating test.

Fig.2

A) Upon induction of apoptosis with TNFα, the death receptor DR6 forms a complex with PSAP. HeLa cells cultured in the presence of 10 ng/ml TNFα together with 10 μg/ml cycloheximide for 6 h were harvested, washed once in PBS and homogenized in IP buffer followed by to co-immunoprecipitation. Bax (B) and Bak (C), but not Bid, tBid, Bmf, Bim, and Puma, were co-immunoprecipitated with PSAP by all five PSAP-specific antibodies, which recognize different areas of PSAP. D) TNFα induced Bax translocation and cytochrome c release.

DR6 induces PSAP-Bax and DR6-Bax complexes formation

To determine the possible role of PSAP in DR6-regulated apoptosis, cells were transfected with a DR6 expression plasmid. As shown in Fig. 3A, after 24 h transfection, both DR6 and Bax were co-immunoprecipitated with PSAP. In addition, DR6 was also co-immunoprecipitated with Bax (Fig. 3B). These results indicate that, in addition to interacting with PSAP, DR6 induces PSAP-Bax and DR6-Bax complexes formation. The observation that DR6 itself also forms a complex with Bax prompted us to determine whether PSAP plays a role in mediating DR6-Bax complex formation or if DR6 interacts with Bax without the involvement of PSAP. Using the DR6-inducible expression cells, we determined the effects of knockdown of PSAP on the formation of DR6-Bax complex. As shown in Fig. 3C, upon induction of DR6 expression by addition of Tetracycline, DR6-Bax complex was detected in cells treated with non-silencing control siRNA (lane 4) as well as in cells treated with PSAP-specific siRNA (lane 6). This result indicates that DR6 forms a complex with Bax independent of PSAP. Similarly, as shown in Fig. 3D, DR6-PSAP complex was detected in cells treated with Bax-specific siRNA (lane 6), indicating that DR6 forms a complex with PSAP independent of Bax. It was also noted that similar to PSAP (Fig. 2), DR6 did not form a complex with Bid (line 10 of Fig. 3C, D).

Fig.3

DR6 induces DR6-PSAP, PSAP-Bax, and DR6-Bax complexes formation. A) PSAP forms complex with both DR6 and Bax and (B) Bax forms complex with DR6 upon transfection of HeLa cells with DR6. C) Knockdown of PSAP has no effect on DR6-Bax complex formation. D) Knockdown of Bax has no effect on PSAP-DR6 complex formation. In C and D, HeLa cells treated with or without PSAP- or Bax-specific, or non-silencing siRNA for 48 h were induced for DR6 expression. Twenty fours later, cell lysates were immunoprecipitated with anti-PSAP, anti-Bax antibody or IgG.

PSAP and DR6 interact with Bax in different subcellular locations

To examine the subcellular location of the DR6-Bax, DR6-PSAP, and PSAP-Bax complexes formed upon induction of apoptosis by DR6, after induction of DR6 expression, cell lysates were separated into cytosolic and mitochondrial fractions using “Mitochondria Isolation Kit” from Thermo Scientific. As shown in Fig. 4A, the PSAP-Bax complex was detected in the mitochondrial fraction (lane 6), but not in the cytosol (lane 4). In contrast, DR6-Bax complex was detected in the cytosolic fraction, which contains low-density membranes including microsomes [25] (Fig. 4B, top panel, lane 6); however, this DR6-Bax complex was hardly detected in the mitochondrial fraction (Fig. 4B, middle panel, line 6). Interestingly, the DR6-PSAP complex was not detected in the cytosol (Fig. 4B, middle panel, lane 4), but was detected in the mitochondrial fraction, albeit at a low level (Fig. 4B, middle panel, lane 4). On the other hand, both DR6-PSAP and DR6-Bax complex was detected in the pellet fraction, which contains the unbroken cells, large membrane fragments, and cell debris [26] (Fig. 4B, bottom panel, lanes 4 and 6). These results indicate that the DR6-PSAP, DR6-Bax, and PSAP-Bax complexes are localized in different intracellular compartments. Next, we determined the role of DR6 in TNFα-induced PSAP-Bax complex formation. As shown in Fig. 4C, efficient knockdown of DR6 was confirmed by western blot analysis (compare lanes 3 and 4 with lanes 1 and 2). It was noted that knockdown of DR6 had no significant effect on TNFα-induced apoptosis as determined by the cleavage of PARP (middle panel). This is likely because TNFα-induced apoptosis is not solely mediated by DR6. However, as shown in Fig. 4D and 4E, the level of PSAP-Bax complex was detected at a significantly lower level in DR6-knockdown cells compared with that of non-silencing siRNA-treated cells (compare lane 6 with lane 4). This result suggests that DR6 plays a crucial role in TNFα-induced PSAP-Bax complex formation.

Fig.4

DR6-induced complexes formed at different subcellular location. A, B) After 24 h induction of DR6 expression, cell lysates were separated into cytosolic, mitochondrial, and pellet fractions and then immunoprecipitation was carried. A) Bax was co-immunoprecipitated with PSAP from mitochondrial fraction (lane 6), but not cytosolic fraction (lane 4). B) Bax (lane 6), but PSAP (lane 4) was co-immunoprecipitated with DR6 from cytosol (top panel); PSAP (lane 4), but not Bax (lane 6) was co-immunoprecipitated with DR6 from mitochondria (middle panel); DR6 was co-immunoprecipitated with both PSAP (lane 4) and Bax (lane 6) from pellet fraction (bottom panel). C) Knockdown of DR6 has no significant effect on TNFα induced apoptosis (middle panel lane 4). Efficient knockdown of DR6 was confirmed by western blot (top panel lanes 3 and 4). D) Knockdown of DR6 strongly reduced TNFα-induced PSAP-Bax complex formation (compare lane 6 with lane 4). E) Densitometric analysis was performed on representative blots from three individual experiments (Fig. 4D), values normalized, and data analyzed by ANOVA with PRISM statistical analysis software. Results (mean±SEM) are shown and the difference between control and DR6 siRNA-treated cells was significant (**p < 0.01).

PSAP is required for DR6-induced apoptosis

To further determine the role of PSAP in DR6-induced apoptosis, we next examined the effect of knockdown of PSAP on DR6-induced apoptosis. As shown in Fig. 5A, in contrast to control siRNA, treatment with PSAP-specific siRNA strongly blocked DR6-induced PARP cleavage and caspase activation (compare lane 4 with lane 2), indicating that knockdown of PSAP strongly inhibited DR6-induced apoptosis. As a control, transfection with LacZ protein did not cause any apoptotic responses in cells treated with either control (non-silencing) siRNA (lane 1) or PSAP-specific siRNA (lane 3). Our previous study showed that DR6-induced apoptosis involves Bax translocation and cytochrome c release [9]. We next examined the effect of knockdown of PSAP on DR6-induced Bax and Cytochrome c relocation. As shown in Fig. 5B, in the control siRNA treated cells, transfection with DR6 resulted in Bax translocation from the cytosol to the mitochondria (second panel, lanes 2 and 6). Concomitantly, cytochrome c was released from the mitochondria to the cytosol (first panel, lanes 2 and 6). Similar to cytochrome c, the other mitochondrial apoptotic factor Smac/DIABLO was also released from the mitochondria to the cytosol upon transfection with DR6 (third panel, lanes 2 and 6). In contrast to control siRNA, when cells were treated with PSAP-specific siRNA, neither cytochrome c nor Smac release was detected in the cytosol (first and third panel, lanes 4 and 8). Bax translocation to mitochondria was also inhibited by knockdown of PSAP (second panel, lanes 4 and 8). These data strongly suggest that DR6-induced apoptosis is mediated by PSAP-dependent Bax translocation and the mitochondrial apoptotic factors, cytochrome c and Smac, release.

Fig.5

PSAP is required for DR6-induced cytochrome c release and apoptosis. A) 48 h after treatment with non-silencing siRNA (lanes 1 and 2) or siRNA specific to PSAP (lanes 3 and 4), cells were transiently transfected with either LacZ (lanes 1 and 3) or DR6 (lanes 2 and 4). Knockdown of PSAP almost completely inhibited DR6-induced apoptosis (lane 4). PSAP (top panel), PARP cleavage (second panel), DR6-myc (third panel), caspase activation (panels 4–6), and actin (panel 7) were determined by western blot. B) Cells were transiently transfected with either LacZ or DR6 for 24 h. Cytosolic fraction (left column) and mitochondria-containing membrane fraction (right column) was prepared as described under “Experimental Procedures.” Top panel, release of cytochrome c from the mitochondria to the cytosol; panel 2, translocation of Bax from the cytosol to the mitochondria; panel 3, release of Smac/DIABLO from the mitochondria to the cytosol; panel 4, reprobe of the top panel with anti-COX I antibody, confirming the integrity of mitochondria during preparation.

DISCUSSION

In the current study, in an effort to determine the apoptotic cascades that involve PSAP, our yeast-two hybrid screen of a brain cDNA library led to the identification of the death receptor DR6 as a PSAP-interacting protein. The interaction of PSAP with DR6 was further confirmed by co-immunoprecipitation assays. These findings prompted us to determine the roles of PSAP in DR6-mediated apoptosis. To this end, using siRNA knockdown strategy and co-immunoprecipitation approach, our data demonstrated that PSAP interacts with both DR6 and Bax and that knockdown of PSAP strongly inhibited DR6-induced apoptosis. These results strongly suggest that PSAP is essential for DR6-induced mitochondrial-dependent apoptosis.

DR6 is an orphan receptor of the TNF receptor super family (TNFRSF). Among the known death receptors, DR6 is a unique member, as it was shown that DR6 induces apoptosis through a mechanism independent of the fas-associated protein with death domain (FADD) [24], which is a key molecule transmitting extrinsic apoptotic and necroptotic signals mediated by the conventional death receptors [27]. Our previous study also showed that DR6-induced apoptosis is independent of caspase-8, which is a key enzyme in mediating typical death receptor-induced apoptosis [9]. Our study further revealed that DR6 induces apoptosis dependent of Bax translocation and cytochrome c release and that the relocation of Bax and cytochrome c is independent of tBid formation [9]. These findings strongly suggest that unlike other conventional death receptors, DR6 induces apoptosis through a unique mitochondrial pathway dependent on Bax translocation to the mitochondria [9]. However, it is not clear how DR6 elicits Bax translocation and the subsequent cytochrome c release and apoptosis. In this regard, it is very interesting to note that our current data demonstrated that DR6 interacts with a novel mitochondrial proapoptotic protein PSAP. This finding provides for the first time a direct molecular linkage of DR6 with the mitochondria and suggests that PSAP may play a role in DR6-induced apoptosis.

To explore the possible role of PSAP in DR6-induced apoptosis, our data showed that both DR6 and PSAP forms a complex with Bax upon induction of apoptosis by DR6. Our data further revealed that knockdown of PSAP has no effect on DR6-Bax complex formation. However, knockdown of PSAP strongly inhibited DR6 induced Bax translocation and apoptosis. These observations indicate that, in the absence of PSAP, interaction of DR6 with Bax is not sufficient for DR6 to induce apoptosis, suggesting a role for PSAP in mediating DR6-induced Bax translocation.

Regarding the mechanism by which PSAP mediates DR6 induced Bax translocation, it is noteworthy that DR6-PSAP, DR6-Bax, and PSAP-Bax complexes were detected in different subcellular locations. The fact that the DR6-Bax complex was detected in the cytosolic fraction but not in the mitochondrial fraction, while on the other hand the PSAP-Bax complex was detected in the mitochondrial fraction but not in the cytosolic fraction, suggests that DR6-bound Bax is possibly transferred to PSAP upon interaction with PSAP at the mitochondria and that PSAP functions as a receptor or anchor for Bax translocation to the mitochondria. This notion is supported by the finding that DR6-Bax complex formation occurs independently of PSAP; however, knockdown of DR6 significantly reduced the level of PSAP-Bax complex in the mitochondria upon treatment with TNFα. Mitochondrial targeting of Bax is a key step in Bcl-2 protein regulated mitochondrial apoptotic pathway. However, the mechanism of Bax translocation is still not well understood. Specifically, the molecule(s) that function as a receptor for Bax on mitochondrial membrane has not been identified [28]. Thus, the observation that PSAP may function as a receptor for DR6 induced Bax translocation provides a new model system to study mechanisms of activation and targeting Bax to the mitochondria. It was noted that PSAP could induce apoptosis independent of Bax and Bak [8]. However, this does not contradict with our current finding, because it cannot be ruled out that PSAP may function as an anchor for Bax and/or Bak in circumstances such as DR6 regulated apoptosis. On the other hand, the putative six transmembrane domains structure makes PSAP capable of forming a channel on the mitochondrial membrane. Given the fact that Bax induces cytochrome c release by multiple mechanisms, including the opening of existing channels [29], it would be interesting to investigate whether PSAP functions as a channel protein in future studies.

DR6 is a relatively a new member of the death receptor family. The DR6 apoptotic pathway has been implicated in the regulation of many cellular events including axonal growth during development and neuronal cell death observed in neurodegenerative diseases, such as amyotrophic lateral sclerosis, AD, and prion disease. In addition, DR6 has been shown to control proliferation and differentiation of immune cells and contribute to autoimmunity. Despite the important roles of DR6 in many different systems, the intracellular molecular cascades downstream of DR6 remain largely unknown. The findings of this study that DR6 directly interacts with a novel mitochondrial proapoptotic protein PSAP shed an interesting new light on the mechanism by which DR6-induces apoptosis through a unique mitochondrial pathway. Given the fact that PSAP is specifically expressed in the mitochondrial outer membrane [6], our data also strongly suggest that PSAP functions as a receptor to anchor Bax on the mitochondrial outer membrane. This notion suggests a unique model of death receptor mediated mitochondrial apoptotic pathways. In this regard, it is notable that PSAP is a homologue of mitochondrial carrier protein and, thus, also known as Mtch1. The other member of this family, Mtch2 has also been implicated in apoptosis by recruiting the pro-apoptotic Bcl-2 family member tBid to mitochondria [30]. However, in contrast to Mtch2, which specifically binds to tBid, PSAP (Mtch1) specifically interacts with Bax/Bak. Nevertheless, these observations support the notion that mitochondrial carrier homolog family proteins play an important role in mitochondrial apoptotic pathway. Specifically, our data strongly suggest that PSAP functions as an anchor or target for DR6 activated Bax on the mitochondria. Results of previous studies on the contribution of DR6 to the development of AD have been controversial [11, 31]. It is notable that this inconsistency may be due to an inappropriate mouse model was used in the second study as pointed out by Dr. Marc Tessier-Lavigne, who is a co-author on both of the articles, in a forum discussion [32]. Our results established for the first time a molecular link between DR6 and the mitochondrial apoptotic cascade. This finding may open a new avenue to determine the pathological function of DR6 and the mechanism by which DR6 is involved in neurodegenerative diseases including AD and amyotrophic lateral sclerosis.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China No. 81701076 (to Linlin Zeng) and No. 31670795 (to Xueqi Fu), Changbai Mountain Research Support Foundation of 2017 (No. 440050117010, to Xueqi Fu), by Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences YKFJ2-007 (to Linlin Zeng), by National Institutes of Health Grants NS095256 (to X. X.) and HL107466 (to M.-Z. C.). This work was also supported by an Alzheimer’s Association grant and a grant from the American Health Assistance Foundation (to X. X.)

We thank Jay English and Susan Perez for critical reading of the manuscript.

Authors’ disclosures available online ().

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