InteractiveFly: GeneBrief

Defender against apoptotic cell death 1: Biological Overview | References

How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. This study found that functional loss of a gene in Drosophila, named Drosophila defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model (Zhang, 2016).

Cell apoptosis, or programmed cell death (PCD), is one of the most fundamental processes essential for normal development in multicellular organisms. Cell apoptosis is tightly controlled to eliminate excess or damaged cells during development, while dysregulation can lead to pathological diseases, such as neurodegenerative diseases and cancer. Although regulation of cell apoptosis involves a sophisticated and complex molecular cascade, the core machinery that initiates, mediates and executes programmed cell death as well as related regulatory pathways are evolutionary conserved among species. Drosophila melanogaster, with its well-annotated genome and established genetic tools, is commonly used to study mechanism of cell apoptosis and discover novel regulatory pathways and genes for regulating development and tissue homeostasis (Zhang, 2016).

The defender against apoptotic cell death 1 (Dad1) gene was first identified and considered as a negative regulator of cell apoptosis from a temperature-sensitive hamster cell line, tsBN7. With the temperature sensitive mutation in Dad1 gene, cells are normal at permissive temperature, while shifting to restrictive temperature led to programmed cell death. Homologous proteins of hamster Dad1 from other species were found to be highly conserved in sequence and function. The yeast homologue of Dad1 is called Ost2 with 40% identity compared to hamster Dad1 and ost2 mutant induces yeast cell apoptosis. The function of Dad1 has been studied in multicellular organisms as well. Mouse Dad1 mutants exhibited developmental delay, aberrant morphology and increased cell apoptosis during embryogenesis and only survived up to stage of midgestation. In Caenorhabditis elegans, overexpression of either Caenorhabditis Dad1 or human Dad1 induced by a heat-shock promoter inhibited developmental cell death during embryogenesis. While ectopic expression of Dad1 in mouse thymocytes did not increase survival of T cells under apoptotic stimuli, peripheral T cells from spleen and lymph nodes in Dad1 transgenic mice increased cell proliferation. These results indicate that Dad1 plays a critical role in regulating cell viability and apoptosis (Zhang, 2016).

Dad1 is identified as a subunit of oligosaccharyltransferase complex (OST), which acts at the first step for protein N-linked glycosylation. N-linked glycosylation is an important protein modification process, which transfers oligosaccharide to selected asparagine of target nascent proteins to ensure their proper folding and maturation in the endoplasmic reticulum (ER). In eukaryotes, OST complex has a catalytic subunit Stt3 as a core and other six non-catalytic subunits, including Ribophorin I, Ribophorin II, Ost48, Dad1/Ost2, N33/Ost3, and Ost4. There are two OST catalytic subunit genes in vertebrates and insects, referred to as Stt3A and Stt3B. Non-catalytic subunits facilitate the catalytic function of Stt3A and Stt3B. Among them, Ost48 and Dad1 are critical for assembly and stability of Stt3 as well as the OST complex, and loss of them can lead to hypoglycosylation (Roboti, 2012). Ribophorin I escorts the N-glycosylation of OST complex on specific substrates, especially some membrane proteins. Because loss of any one non-catalytic subunit does not fully abolish the function of OST, it is possible that they may have redundant roles or different non-catalytic subunits assist the glycosylation of specific targets (Zhang, 2016).

Dad1, as an N-glycosylation regulatory protein, plays an important role in cell survival, but up to now no detailed mechanism is known about how loss of Dad1 induces cell apoptosis. Based on the evidence from studies in different organisms, Dad1 may facilitate the OST complex to target specific proteins that directly maintain cell survival. Another possibility is that accumulation of unfolded or misfolded proteins of hypoglycosylation at ER triggers stress signaling and initiates programmed cell death. It is also possible that Dad1 affects cell viability in an OST-independent manner. For instance, Dad1 interacts with a Bcl2 family protein, Mcl1, which might lead to apoptosis inhibition (Zhang, 2016).

This study used Drosophila Dad1 (dDad1), which shows more than 70% identity with human DAD1, to study how loss of Dad1 function affects cell viability and tissue growth. Loss of Dad1 in Drosophila was found to decreased tissue growth due to induction of apoptosis and absence of cell proliferation. dDad1 is required for efficient N-glycosylation in developing tissues and the small wing phenotype induced by dDad1 knock-down can be enhanced by reducing gene dosage of the OST catalytic subunit. In addition, the c-Jun N-terminal kinase (JNK) pathway was found to mediate dDad1 knockdown-induced cell apoptosis in wing discs through mitogen-activated protein kinase kinase kinase 1 (Mekk1) and wallenda (wnd). Moreover, in dDad1 knock-down tissues, ER stress is induced and Perk-Atf4 pathway functions upstream of the JNK pathway. Intriguingly, unfolded protein response (UPR) signaling appears to play a dual role in inducing cell death and stimulating compensatory proliferation of neighboring cells for tissue homeostasis (Zhang, 2016).

This study found that loss of dDad1 in the wing interferes with N-linked protein glycosylation, which triggers ER stress and activates the downstream JNK pathway to cause apoptosis. Interestingly, although this study suggests that dDad1 regulates tissue growth through the OST complex, only one of two catalytic subunits, Stt3A (CG1518), genetically interacts with dDad1 in growth control. With the association of dDad1, Stt3A may become the major functioning complex modifying crucial proteins or more nascent proteins in developing tissues, therefore inducing severe or prolonged ER stress if dDad1 activity is absent. Different tissues or regions of tissues may have different levels of sensitivity to the loss of dDad1, which could explain why cell apoptosis was more frequently observed in wing pouch than other region of the wing disc in these experiments. Similar phenomena were also reported in other studies. For instance, Dad1 mutant mice exhibit cell death which leads to developmental defect and embryonic lethality, but the death of cells only happens in some tissues of the embryo (Zhang, 2016).

One study reports that dDad1 homozygous mutant clones in larval fact body induced cell autophagy (Arsham, 2009). However, the current study did not observe cell autophagy using lyso-tracker staining in dDad1 mutant clones in larval wing discs. The different response may be due to different tissue-specific context. Therefore, apoptosis induced by loss of dDad1 in the wing is unlikely to be dependent of autophagy process (Zhang, 2016).

As an N-glycosylation regulator, dDad1 may have other target proteins essential for animal viability. In this study, although blocking Perk-Atf4 or JNK pathway suppresses apoptosis induced by loss of dDad1, the lethality of en-Gal4/UAS-dDad1 RNAi individuals cannot be rescued. Thus, cell apoptosis induced by loss of dDad1 may not be the major reason for causing lethality of the mutant organism. It would be interesting to identify potential targets of dDad1 that are essential for normal development of Drosophila (Zhang, 2016).

The Perk-Atf4 pathway may play both protective and destructive roles in growth control in loss of dDad1 model: on one hand, it activates JNK signaling to cause cell apoptosis; on the other, it may send out signals to neighboring cells to increase cell proliferation (Zhang, 2016).

In Drosophila, it is known that prolonged ER stress induces cell apoptosis, however the mechanism of ER stress activates apoptosis remains elusive. A few Drosophila ER stress models have been established and their connection with apoptosis has been validated. Nevertheless, they suggested different pathways leading to cell death and none of them is the same as what was found in the current study. In a chronic ER stress model due to Presenilin (Psn) overexpression, cell apoptosis is induced in a Perk-Atf4 dependent but JNK-independent manner. Although the study found that the JNK pathway is activated by Perk-Atf4, it induces dilp8 expression which extends developmental stage to keep tissue homeostasis, rather than stimulates cell death. In the current study, induction of cell apoptosis in the absence of dDad1 function was found to be dependent on both Perk-Atf4 and JNK pathways. The JNK pathway acts downstream of Perk-Atf4 and causes cell apoptosis. It was also found that Mekk1 and Wnd are two major JNKKK's that activate JNK pathway in response to loss-of-dDad1 induced ER stress. This is consistent with the observations from another ER stress model, autosomal dominant retinitis pigmentosa (ADRP) model. However, ADRP model suggests that activation of JNK pathway and cell apoptosis does not rely on UPR signaling. In mammalian models of ER stress, cell apoptosis is induced through activation of IRE1-XBP1 branch, followed by JNK pathway activation via TRAF2/ASK1. In the current study, it was not possible to test the role of Ire1-Xbp1 branch due to the embryonic lethality caused by expression of Ire1 RNAi or Xbp1 RNAi driven by en-Gal4, although its activation was observed. Ask1 does not seem to play a role in mediating cell apoptosis in the loss-of-dDad1 model, since knocking down of Ask1 cannot restore loss-of-dDad1 induced wing size deduction (Zhang, 2016).

Compensatory proliferation is a self-rescue mechanism to keep tissue homeostasis when some cells are under stress or apoptosis. Mechanism of compensatory proliferation was mostly studied in Drosophila. In fly wing discs, Dronc in the apoptotic cells stimulates JNK pathway, inducing expression of mitogens, Dpp and Wg, to presumably promote proliferation of neighboring cells. Interestingly, in this study, activation of Perk-Atf4 pathway appears to play a role in inducing extra cell proliferation but does not rely on activation of the JNK pathway nor cell death. It suggests that Atf4 transcription factor may induce transcription of mitogenic genes to stimulate cell proliferation, in addition to the genes that are related to stress response (Zhang, 2016).

N-glycosylation of Wg was affected in dDad1 mutant cells. However, it turned out that N-glycosylation of Wg is unimportant for its secretion and signaling in fly tissues. Regardless, it is unlikely that Wg was responsible for mediating extra cell proliferation in this loss-of-dDad1 model based on the following observations. Firstly, compensatory cell proliferation can still occur in the absence of wg and dpp in tissues undergoing massive cell apoptosis. Moreover, this study found that proliferation of extra cells can still occur when JNK signaling was blocked, while the JNK pathway is presumably responsible for Wg induction. Therefore, further studies are needed to address how Perk-Atf4 signaling acts to induce compensatory cell proliferation to maintain tissue homeostasis (Zhang, 2016).

Some studies suggest that Dad1 may have a direct effect on cell death suppression. Overexpression of hDAD1 or ceDAD1 has been shown to be sufficient to inhibit developmental cell apoptosis in C. elegans. Overexpression of DAD1 in mouse thymocyte did not increase survival of T cells under apoptosis stimuli, however peripheral T cells from spleen and lymph node in Dad1 transgenic mice have increased cell proliferation. This work examined the phenotypes of dDad1 transgenic flies in different tissues. Drosophila pupa eye is a good model to study developmental apoptosis because wild-type flies have fixed number of interommatidial cells in mid-pupal eye discs due to the elimination of excess cells by apoptosis. This study found that overexpression of dDad1 in the eye did not cause the accumulation of extra interommatidial cells in mid-pupal eye discs, which indicates that dDad1 is not sufficient to block normal apoptosis during eye development. In addition, this study overexpressed apoptosis-inducing genes in Drosophila wing and eye to test if dDad1 is able to suppress induced apoptosis. The cell apoptosis in the wing induced by overexpression of UAS-eiger failed to be suppressed by dDad1 overexpression. However, overexpression of dDad1 could moderately rescue cell death induced by GMR-hid in adult eyes. These observations indicate that Drosophila dDad1 inhibits induced cell apoptosis moderately and conditionally, but exhibits no effect on normal developmental apoptosis. Therefore, these observations suggest that dDad1 may not function as an active defender of cell apoptosis in Drosophila (Zhang, 2016).

Search PubMed for articles about Drosophila Dad1

Arsham, A. M. and Neufeld, T. P. (2009). A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway. PLoS One 4(6): e6068. PubMed ID: 19562034

Roboti, P. and High, S. (2012). The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation. J Cell Sci 125(Pt 14): 3474-3484. PubMed ID: 22467853

Zhang, Y., Cui, C. and Lai, Z. C. (2016). The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 27693235

date revised: 12 December 2018

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