InteractiveFly: GeneBrief

Mesencephalic astrocyte-derived neurotrophic factor: Biological Overview | References

Gene name - Mesencephalic astrocyte-derived neurotrophic factor

Synonyms -

Cytological map position - 89B13-89B13

Function - secreted

Keywords - unfolded protein response, immune modulation, expressed in glial cells - supports the dopaminergic system in non-cell-autonomous manner

Symbol - Manf

FlyBase ID: FBgn0027095

Genetic map position - chr3R:16,349,404-16,350,801

NCBI classification - Armet: Degradation arginine-rich protein for mis-folding

Cellular location - secreted

NCBI links: Precomputed BLAST | EntrezGene

Regenerative therapies are limited by unfavorable environments in aging and diseased tissues. A promising strategy to improve success is to balance inflammatory and anti-inflammatory signals and enhance endogenous tissue repair mechanisms. This study identified a conserved immune modulatory mechanism that governs the interaction between damaged retinal cells and immune cells to promote tissue repair. In damaged retina of flies and mice, platelet-derived growth factor (PDGF)-like signaling induced mesencephalic astrocyte-derived neurotrophic factor (MANF) in innate immune cells. MANF promoted alternative activation of innate immune cells, enhanced neuroprotection and tissue repair, and improved the success of photoreceptor replacement therapies. Thus, immune modulation is required during tissue repair and regeneration. This approach may improve the efficacy of stem-cell-based regenerative therapies (Neves, 2016).

This study has confirmed that MANF is expressed in fly innate immune cells (hemocytes) using immunohistochemistry of hemolymph smears from late 2nd instar larvae. In these smears, hemocytes were identified by Green Fluorescent Protein (GFP) expression driven by the hemocyte specific driver Hemolectin:Gal4 (HmlΔ:Gal4). MANF was also detected by immuno blot in the plasma fraction of the hemolymph, confirming its secretion. Consistent with the RNAseq data, Reverse Transcription and Real Time quantitative Polymerase Chain Reaction (RT-qPCR) analysis revealed that MANF mRNA levels were significantly higher in hemocytes from UV treated larvae compared to untreated controls, and that this induction was PvR dependent. Over-expression of Pvf-1 in the retina (using GMR:Gal4; Glass Multimer Reporter as a driver) was sufficient to induce MANF mRNA specifically in hemocytes, in the absence of damage, and was accompanied by a significant increase in MANF protein in the hemolymph (Neves, 2016).

Flies overexpressing MANF in hemocytes showed significant tissue preservation after UV exposure, even after PvR knock-down in hemocytes. This protective activity of hemocyte-derived MANF was further confirmed in two genetic models of retinal damage, in which degeneration is induced by retinal (GMR driven) over-expression of the pro-apoptotic gene grim or of mutant Rhodopsin (Rh1G69D) (Neves, 2016).

Null mutations in the manf gene are homozygous lethal at early 1st instar larval stages, yet MANF heterozygotes (which express significantly lower levels of MANF in hemocytes compared to wild-types) had a significantly increased tissue degeneration response to UV. This increase in tissue loss could be rescued by MANF over-expression in hemocytes and was recapitulated by hemocyte-specific knock-down of MANF (Neves, 2016).

The protective effect of hemocyte-derived MANF could be caused by direct neuroprotective activity of MANF on retinal cells, or could reflect an indirect effect of MANF on the microenvironment of the damaged retina. To distinguish between these possibilities, whether MANF could influence hemocyte phenotypes was tested. Hemocytes can acquire lamellocyte phenotypes, characterized by down-regulation of plasmatocyte markers (hemolectin, hemese) and expression of Atilla protein, during sterile wound healing. These phenotypes correlate with hemocyte activation and may influence tissue repair capabilities, and they were recapitulated in the UV damage paradigm. Over-expression of MANF in hemocytes in vivo or treatment of hemocytes in culture with human recombinant MANF (hrMANF) significantly increased the proportion of lamellocytes in hemocyte smears, as detected by Atilla expression. This correlated with a decrease in the proportion of cells expressing GFP driven by HmlΔ:Gal4 and a decrease in hml transcripts. Furthermore, MANF was necessary and sufficient to induce the Drosophila homolog of the mammalian M2 marker arginase1 (arg) in hemocytes, suggesting that these cells may be able to acquire phenotypes similar to alternative activation. Most MANF expressing hemocytes also expressed Arg, suggesting that there is an association between MANF expression and M2-like activation of hemocytes (Neves, 2016).

To test whether MANF's immune modulatory function is required for retinal repair, retinal tissue preservation was assessed in conditions in which hemocytes express and secrete high levels of MANF, but are unable to be activated in response to this signal. Such a condition was generated by overexpressing MANF in the absence of Kdel Receptors (KdelRs). In human cells, KdelRs modulate MANF secretion and cell surface binding. Intracellular KdelR prevents MANF secretion, while cell surface bound KdelR promotes binding of extracellular MANF. Knock-down of the one Drosophila KdelR homologue in hemocytes resulted in a significant induction of MANF transcripts and the detection of MANF protein in the hemolymph, suggesting that KdelR-depleted hemocytes secrete high levels of MANF. In these hemocytes, MANF-induced lamellocyte formation and Arg expression were significantly decreased. Hemocyte activation by extracellular MANF is thus impaired after KdelR knock-down. This genetic perturbation also resulted in a significant enhancement of UV-induced tissue loss, which could not be rescued by MANF over-expression. Thus, immune modulation by MANF is critical for tissue repair (Neves, 2016).

The results identify MANF as an evolutionarily conserved immune modulator that plays a critical role in the regulatory network mediating tissue repair in the retina. The ability of MANF to increase regenerative success in the mouse retina highlights the promise of modulating the immune environment as a strategy to improve regenerative therapies (Neves, 2016).

MANF has previously been described as a neurotrophic factor (Lindholm, 2007; Voutilainen, 2009), and it may also exert a direct neuroprotective effect in the retina, yet the data suggest a more expansive role: because MANF cannot promote tissue repair in flies in which the hemocyte response to MANF is selectively ablated, or in mammalian retinas depleted of innate immune cells or containing macrophages that are unresponsive to MANF, it is proposed that MANF's role in promoting alternative activation of innate immune cells is central to its function in tissue repair. Further studies will be required to determine the specific contribution of alternative-activated macrophages in mediating these effects. While the data point to an important role of macrophages in mediating the effects it does not exclude the possibility that other cell types are involved in the process, nor that macrophages' functions other than polarization may influence the outcome of MANF's protective effects (Neves, 2016).

Clinically, MANF may thus have a distinct advantage over previously described neurotrophic factors in both improving survival of transplanted cells directly, as well as in promoting a microenvironment supportive of local repair and integration. Because integration efficiency correlates with the extent of vision restoration it can be anticipated that MANF supplementation will have an important impact in clinical settings (Neves, 2016).

Further studies involving tissue specific knockdown of MANF in mammals will be required to evaluate the relative contribution of different cellular and tissue sources for MANF in homeostatic and damage conditions. While this study found that MANF is strongly expressed in immune cells, MANF expression was also observed in other cell types, in agreement with previous reports (Neves, 2016).

Similarly, the molecular mechanism involved in MANF signaling remains elusive. To date, a signal transducing receptor for MANF has not been identified, although Protein kinase C (PKC) signaling has been described to be activated downstream of MANF. MANF can further negatively regulate NF-κB signaling in mammalian cells and loss of MANF in Drosophila results in the infiltration of pupal brains with cells resembling hemocytes with high Rel/NFκB activity, potentially representing pro-inflammatory, M1-like phenotypes. The identification of immune cells as a target for MANF in this study may accelerate the discovery of putative MANF receptors and downstream signaling pathways (Neves, 2016).

Because neurotoxic inflammation has been implicated in Parkinson's disease, it is possible that the protective effects of MANF in this context are also mediated by immune modulation, as this study has shown for retinal disease. Indeed, recent reports suggest that the MANF paralog, cerebral dopamine neurotrophic factor (CDNF), has an anti-inflammatory function in murine models of Parkinson's disease and in nerve regeneration after spinal cord injury. A recent study has further shown that loss of MANF leads to beta cell loss in the pancreas. Beta cell loss is a commonly associated with chronic inflammation, and it is thus tempting to speculate that MANF is broadly required in various contexts to aid conversion of pro-inflammatory macrophages into pro-repair anti-inflammatory macrophages. Future studies will clarify the role of MANF in resolving inflammation and promoting tissue repair not only in the retina and brain, but also in other tissues. A deeper understanding of MANF-mediated immune modulation and its impact on stem cell function, wound repair and tissue maintenance is thus expected to help in the development of effective regenerative therapies (Neves, 2016).

Exploring the Conserved role of MANF in the unfolded protein response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. This study discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach, genetic interactions were found between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. These data suggest a role for Manf in the regulation of Drosophila UPR (Lindstrom, 2016).

The accumulation of unfolded or misfolded proteins causes disturbances in endoplasmic reticulum (ER) homeostasis, a phenomenon referred to as ER stress. ER stress in turn activates the unfolded protein response (UPR). In order to overcome ER stress, UPR leads to attenuation of protein synthesis, enhancement of degradation of unfolded proteins, and activation of specific signalling cascades. These events aim to reduce the overall protein load in the ER and to enhance the protein folding capacity by selective transcription of chaperones. UPR is activated through three signalling cascades by ER transmembrane sensor proteins PERK (PRKR-like endoplasmic reticulum kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor 6). All of these three proteins are maintained inactive in normal cellular status by binding to the major ER chaperone GRP78/BiP (Glucose-regulated protein 78/Binding immunoglobulin protein). Upon ER stress, GRP78 is dissociated from the sensor proteins which are subsequently activated. The most ancient of these signalling cascades is mediated by IRE1, the sole branch of UPR identified in Saccharomyces cerevisiae. IRE1 has kinase activity and endoribonuclease activity needed for degradation of mRNAs in order to relieve the protein synthesis load. IRE1 is also responsible for the unconventional splicing and thus activation of transcription factor XBP1 (X-box Binding Protein-1), a positive regulator of ER chaperone and other UPR related gene expression. Activated PERK attenuates overall protein synthesis through phosphorylating and thus inhibiting EIF2α (eukaryotic translation initiation factor 2, subunit 1 α). However, the decreased activation of EIF2α results in an upregulated translation of specific target mRNAs including ATF4 (activating transcription factor 4). The third signalling pathway is mediated through ATF6, a transcription factor activated by its cleavage and translocation to the nucleus (Lindstrom, 2016).

In Drosophila, both IRE1- and PERK-mediated UPR signalling cascades are conserved. The amino acid sequence of the Drosophila homologue of ATF6 is highly similar to its mammalian counterpart, but experimental evidence for its involvement in Drosophila UPR is lacking. Similar to mammals, the expression of Drosophila homologue of GRP78, Hsc3 (Heat shock protein cognate 3), is upregulated upon induced ER stress in Xbp1-dependent manner but no biochemical data are available to show its association with ER stress sensor proteins (Lindstrom, 2016).

The MANF/CDNF family of neurotrophic factors was first characterized based on its trophic function on dopaminergic neurons in vitro and in vivo. When injected into the brain, recombinant mammalian MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) and CDNF (Cerebral Dopamine Neurotrophic Factor) protect and repair dopaminergic neurons in toxin-induced rodent models of Parkinson's disease (PD) in vivo. The sole Drosophila homologue, DmManf, is expressed in and secreted from glial cells and supports the dopaminergic system in non-cell-autonomous manner (Palgi, 2009). The role of MANF as an extracellular trophic factor is further supported by the evidence that mammalian MANF is protective against ischemic injury in both neurons and cardiomyocytes (Airavaara, 2010; Glembotski, 2012). However, the biology of MANF is not thoroughly understood. Intriguingly, MANF localizes to the ER and has a protective role against ER stress in vitro and in vivo. Additionally, mammalian MANF binds GRP78 in Ca2+-dependent manner in vitro and this binding may regulate MANF secretion. MANF can be retained in the ER by its C-terminal signal sequence, RTDL in human and RSEL in Drosophila (Glembotski, 2012; Lindström, 2013). Experimental evidence suggests that mammalian MANF interacts with KDEL-R [KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor] and that the C-terminal RTDL sequence of MANF is responsible for this interaction. The relevance of KDEL-R as a mediator of the functions of MANF has not been explored in vivo, yet. Recently, MANF was also shown to regulate the expression of ER-resident protein CRELD2 (Lindstrom, 2016).

Both in vivo and in vitro studies have shown that MANF is upregulated after chemically induced ER stress and by misfolded mutant proteins accumulating in the ER. Mammalian MANF expression is activated upon ER stress by ATF6 and XBP1 through an ER stress response element II found in the promoter region of MANF. Based on a knockout mouse model, MANF was found to be essential for the survival of pancreatic β-cells and its loss resulted in severe diabetes due to reduction of beta cell mass and activation of UPR in the pancreatic islets. The protective role against 6-OHDA induced and ischemic neuronal damage has been suggested to rise from the ER-related functions of MANF as these processes have been shown to induce ER stress (Lindstrom, 2016).

In Drosophila, the loss of DmManf is associated with upregulated expression of genes involved in UPR (Palgi, 2012). Additionally, the overexpression of DmManf resulted in downregulation of several UPR-related genes (Palgi, 2012). This study shows that, similar to mammalian MANF, the expression of DmManf is induced in response to ER stress in vitro. Further, transgenic approaches for gene silencing in vivo were applied to reveal genetic interactions between DmManf and genes with known functions in the maintenance of ER homeostasis and in UPR (Lindstrom, 2016).

Increasing evidence indicates that ER stress and UPR play a major role in variety of human diseases including diabetes mellitus and neurodegenerative disorders. MANF is a secreted protein (Palgi, 2009; Lindholm, 2008), but also localizes to the ER and has a role in mammalian UPR (Mizobuchi, 2007; Palgi, 2012). This study examined the role of DmManf in UPR in the Drosophila model. Upregulation of MANF mRNA expression by ER stress-inducing agents was shown to be conserved in Drosophila S2 cells. Additionally, genetic interaction between DmManf and genes known to function in the ER and UPR were shown (see A simplified presentation of UPR and genetic interactions discovered for Drosophila Manf; Lindstrom, 2016).

One of the interacting partners was Hsc3, the Drosophila homologue of mammalian chaperone GRP78. The silencing of Hsc3 in the wing resulted in an abnormal wing phenotype in wild type background. This wing phenotype was stronger in DmManf-overexpressing background. In cultured mammalian cells MANF has been shown to bind GRP78 in Ca2+-dependent manner and the loss of interaction between mammalian MANF and GRP78 was associated with increased secretion of MANF (Glembotski, 2012). In line, the knockdown of Hsc3 could lead to increased secretion of DmManf and lead to deprivation of intracellular DmManf. In a previous study, it was noticed that the deletion of ER retention signal RSEL increased the secretion of DmManf in S2 cells and decreased its functionality in rescue experiments in vivo (Lindström, 2013). Based on the physical interaction found between mammalian MANF and GRP78, the simultaneous overexpression of DmManf and knockdown of Hsc3 could also result in the abundant DmManf binding the residual Hsc3 and preventing other important cellular functions of Hsc3. Alternatively, the loss of Hsc3 could lead to decreased protein folding capacity in the ER and activation of UPR. The vast amount of DmManf protein could exhaust this already disturbed cellular state (Lindstrom, 2016).

In previous studies, mammalian MANF has been suggested to have chaperone-like functions, e.g. by binding unfolded proteins in vitro but the putative chaperone activity remains unconfirmed. The major ER chaperone Hsc3 and DmManf clearly have distinct roles as either the overexpression or the loss of one could not complement for the loss of the other. However, the current study indicates that the interaction between MANF and GRP78 (Glembotski, 2012) is conserved. In future, the functional significance of this intriguing interaction deserves to be addressed in detail (Lindstrom, 2016).

DmManf genetically interacted with PEK/PERK, an ER stress sensor protein. Similar to the silencing of Hsc3, simultaneous overexpression of DmManf worsened the phenotypes observed in PEK knockdown flies. Previous studies have indicated functional conservation of PERK in Drosophila and mammals. The Drosophila homologue to ATF4, the downstream target of activated PERK and selectively upregulated by UPR, showed no genetic interaction with DmManf in this study. It was previously shown that the abolishment of both zygotic and maternal DmManf resulted in increased phosphorylation of eIF2α, another molecular marker used for detecting ER stress (Palgi, 2012). This study abolished only the zygotic DmManf while maternal DmManf was still present. The loss of zygotic DmManf alone did not induce UPR when evaluated by other readouts, i.e. increased Hsc3 mRNA level and splicing of Xbp1. Although the zygotic DmManfΔ96 mutant larvae show only low amount of persisting maternal DmManf mRNA and protein, it could be sufficient to prevent the induction of UPRl (Lindstrom, 2016).

Additionally, a genetic interaction was discovered between DmManf and Xbp1, a transcription factor mainly responsible for the regulation of UPR-induced genes. Upon UPR, the mRNA of Xbp1 is spliced by IRE1 and translated into a transcriptional activator of chaperone expression in response to the increased protein folding demand. According to previous studies, the spliced form of Xbp1 could mediate the UPR-induced upregulation of MANF in mammals. MANF has been suggested to have protective role against ER stress. During normal development, ER stress is detected in the secretory cells and the silencing of Xbp1 disturbs this developmental ER stress. Both mammalian and Drosophila MANF has been shown to have especially high expression levels in secretory tissues. Overexpression of DmManf increased Xbp1 mRNA level but the knockdown of Xbp1 did not affect DmManf expression levels. Also, the mRNA levels of Hsc3 were not upregulated in Xbp1 knockdown larvae. This could indicate the lack of transcriptional activation of DmManf and Hsc3 expression by Xbp1s in Xbp1-knockdown larvae. Therefore, knockdown of Xbp1 could compromise the regulation of DmManf expression in the developmental ER stress and deteriorate its function in the secretory cells (Lindstrom, 2016).

ERAD is a cellular process aiming to clear out the unfolded and misfolded proteins from the ER. According to previous transcriptome analysis, sip3 was downregulated in DmManfΔ96 mutant larvae (Palgi, 2012). This study also found a genetic interaction between DmManf and sip3. Sip3 encodes a homologue to mammalian ER resident E3 ubiquitin ligase synoviolin/HRD1 with specific function in ERAD. Mammalian MANF is upregulated by ERSE-II (ER stress response element II) found in its promoter region. Interestingly, ERSE-II is also found in ERAD-related components HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1, also known as HERP) and VIMP (VCP-interacting membrane protein, also known as selenoprotein S). ERSE-II has been hypothesized to regulate the protein quality control and degradation of misfolded proteins during ER stress suggesting that MANF could also have a role in these functions (Lindstrom, 2016).

Surprisingly, overexpression of DmManf led to enhanced phenotypes in flies of which a UPR-related gene was knocked down. Thus far, overexpression of DmManf with any GAL4 driver tested has never resulted in a detectable phenotype or altered viability. According to a previous microarray analysis, DmManf overexpression led to downregulation of UPR-related genes (Palgi, 2012). This suggests that the overexpression of DmManf would disturb UPR signalling. Hypothetically, wild type background cells would be able to deal with the increased DmManf expression and the subsequent downregulation of UPR-related genes whereas the additional knockdown of an important component of UPR, e.g. Hsc3, PEK or Xbp1, could compromise the cell homeostasis (Lindstrom, 2016).

An alternative explanation for these observations in interaction studies between UPR genes and DmManf would be that DmManf is actually a substrate for UPR. Then, the abundant expression of DmManf by UAS/GAL4 would rather model the effects of increased overall protein synthesis in ER than indicate specific ER-related functions for DmManf. DmManf enters the secretory pathway (Palgi, 2009) and its ectopic expression may cause stress to the protein folding machinery in the ER. Although the Xbp1 mRNA level was increased, the expression of Hsc3 was not altered indicating that overexpression of DmManf induces mild UPR. However, no similar effects were seen with overexpression of membrane-directed GFP suggesting that the observed phenomena were specific for DmManf (Lindstrom, 2016).

The previous microarray study found that the total loss of DmManf is associated with upregulated expression of genes involved in UPR (Palgi, 2012). However, in the current study the mRNA levels of Hsc3 and Xbp1 were mildly decreased in DmManf mutant larvae. In the previous study, transcriptome analysis was done from the embryonic DmManf mutants lacking both maternal and zygotic DmManf. In the current study, RNA was collected from zygotic DmManf mutants with the persisting maternal DmManf mRNA and protein. The maternal DmManf is apparently sufficient to prevent induction of UPR and upregulation of UPR related genes (Lindstrom, 2016).

This work provides evidence for the contribution of DmManf in Drosophila UPR. Further biochemical studies on the interaction between DmManf and UPR genes in Drosophila are needed to elucidate the details of this process (Lindstrom, 2016).

Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor that supports and protects dopaminergic neurons. The Drosophila MANF (DmMANF) null mutant animals die during early development, and DmMANF is required for the maintenance of dopamine positive neurites. The aim of this study was to investigate the role of DmMANF during later developmental stages. DmMANF expression in the adult brain is much wider than in the embryonic and larval stages. It is expressed in both glia and neurons including dopaminergic neurons. Clonal analysis showed that DmMANF is not required cell-autonomously for the differentiation of either glia or dopaminergic neurons. In addition, DmMANF overexpression resulted in no apparent abnormal dopaminergic phenotype while DmMANF silencing in glia resulted in prolonged larval stage (Stratoulias, 2015a).

It has been shown that during embryonic stages, DmMANF expression is confined to some glial subpopulations, such as glia surrounding dopaminergic neurons, in longitudinal and in channel glia (Palgi, 2009). This study used immunohistochemistry to identify the cell populations where DmMANF is expressed. In the adult brain, DmMANF is expressed in all glial subtypes, thus having a considerably broader expression pattern compared to embryonic stages. At the subcellular level, DmMANF is localized in all glial processes during the adult, the mid- and late pupal stages. Interestingly, contrary to Drosophila, in rodents MANF is not expressed in glia cells (Stratoulias, 2015a).

The Drosophila optic lobe is very well characterized both anatomically and developmentally; therefore, this study looked in more detail the DmMANF positive glial projections within this area. DmMANF positive glial projections were found to radiate specifically in strata M6 and M7 of the medulla neuropil. Interestingly, dopaminergic neurons exist in the Drosophila optic lobe and project their processes to two layers, M1 and M7. M1 is immediately proximal to the DmMANF positive cell bodies, while M7 is between the strata that DmMANF positive processes radiate. The close proximity of the radiated DmMANF positive glial processes and the dopaminergic neurites in the medulla suggest a possible role of DmMANF in the dopaminergic system (Stratoulias, 2015a).

In rodent brains, MANF is relatively widely expressed in neurons including dopaminergic neurons, but not in glia. In zebrafish, MANF is also expressed in neurons and to a smaller extent also in glia. In Drosophila embryos, however, DmMANF expression has not been detected in any neuronal populations (Palgi, 2009). This study used co-localization studies to investigate whether DmMANF is expressed in adult neurons. In the adult brain, DmMANF does not co-localize with either of the neuronal axonal markers Fas2 or BP104, but it partially co-localizes with the pan-neuronal marker Elav in the neuronal cell somas. Some of these DmMANF positive neurons stain also with the dopaminergic marker TH, suggesting that DmMANF is expressed in dopaminergic neurons. Further analysis revealed that DmMANF is expressed in the cell somas of all seven major dopaminergic clusters of the brain. In addition, and contrary to DmMANF expression in glia, DmMANF was not detected in the processes of dopaminergic neurons, not even when DmMANF was overexpressed under the TH-Gal4 driver. Interestingly, DmMANF overexpression in the dopaminergic neurons resulted in increased TH immunoreactivity. These results show that DmMANF has a dynamic expression pattern during development of the Drosophila nervous system (Stratoulias, 2015a).

DmMANFΔ96 null mutants are larval lethal (Palgi., 2009). Therefore, in order to study the role of DmMANF in the adult brain, techniques such as RNA interference (RNAi) and clonal analysis were used. To create DmMANFΔ96 homozygous mutant clones in the optic lobe, the FRT/FLP technique was used. These clones expressed the pan-glial marker Repo. The MARCM technique was used to see if DmMANF is needed for the survival and differentiation of dopaminergic neurons. The DmMANFΔ96 homozygous mutant clones expressed the dopaminergic marker TH. Based on these results it is concluded that DmMANF is not required in a cell autonomous fashion for the survival and differentiation of either glia or dopaminergic neurons (Stratoulias, 2015a).

MANF has been shown to protect and even rescue the midbrain dopaminergic neurons in vertebrate models of Parkinson's disease. In addition, it has been shown to be overexpressed under ischemic stress as well as in activated microglia. In DmMANF null mutant flies, the dopaminergic neurites are diminished (Palgi, 2009). It was of interest to explore whether DmMANF overexpression has an effect on the dopaminergic system in adults. In all cases, animals developed normally and lived for at least 30 days. The TH positive neurons were quantified in elav-Gal4; UAS-DmMANF, repo-Gal4; UAS-DmMANF and TH-Gal4; UAS-DmMANF 5 day old adult brains and no statistical significant changes were found in the number of dopaminergic neuron somas. This supports the previous observations that DmMANF overexpression does not have apparent phenotypic effects in the nervous system or elsewhere (Stratoulias, 2015a).

Next, DmMANF mRNA was nocked down by employing the RNAi technique coupled with the UAS/Gal4 system. Ubiquitous knockdown of DmMANF using either tub-Gal4 or da-Gal4 resulted into early larval lethality which phenocopies the DmMANFΔ96 null mutant phenotype. To examine if reduced levels of DmMANF in glia has an effect on the adult dopaminergic neurons, repo-Gal4 was used to knockdown DmMANF in all glia. In this case, both male and female animals developed into adults. The TH positive neurons were quantified in repo-Gal4;DmMANF RNAi 5 day old adult brains and as in the case of the UAS-DmMANF overexpression studies, no statistically significant changes were observed in the number of dopaminergic neuron somas. Next, the efficacy of the RNAi effect was enhanced by overexpressing Dicer-2, an approach that is commonly used in Drosophila. However, this led to pupal lethality even when the flies were reared at 18°C. At 26°C, the repo-Gal4; UAS-DmMANFRNAi UAS-Dicer-2 animals died as 3rd instar larvae but remained in this stage for 8 days compared to 2 days of the control (repo-Gal4; UAS-Dicer-2) and wild type larvae. In addition, the repo-Gal4; UAS-DmMANFRNAi UAS-Dicer-2 larvae showed a locomotion phenotype with reduced peristaltic contraction frequency and circular path trajectories, a phenotype that has been attributed to loss of normal postural control due to interneuron inactivation. Furthermore, these animals stayed at the bottom of the tube, they did not climb and they were immobile compared to control and wild-type third-instar larvae (Stratoulias, 2015a).

Next, whether DmMANF knockdown in neurons has an effect on the dopaminergic system was investigated. First, the pan-neuronal driver elav-Gal4 was used to knock down DmMANF in all neurons. Quantification of the TH-positive neurons in adult brains showed no significant differences compared to the wild type brains. Since DmMANF is expressed in dopaminergic neurons, TH-Gal4 was used to knock down DmMANF specifically in these neurons. Again, quantification of the TH-positive neurons showed no significant differences compared to the wild type brains. This result implies that either DmMANF is not required in a cell-autonomous manner to dopaminergic neurons or that TH promoter is not strong enough to silence DmMANF in these neurons. However, sporadic absence of dopamine neuron clusters was noticed in TH-Gal4; UAS-DmMANFRNAi and elav-Gal4; UAS-DmMANF brains. It has earlier been shown that TH-specific expression of Dicer-2 in dopaminergic neurons has a significant effect on motor behavior in Drosophila and possibly also on dopaminergic neuron survival. In accordance with this, dopaminergic phenotypes were seen in TH-Gal4; UAS-Dicer-2 adult brains and therefore this enhancement of RNAi in the dopamine neurons was not used. In summary, further studies are required in order to solve the role of DmMANF in the adult dopaminergic system (Stratoulias, 2015a).

MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain

Glia perform conserved functions not only in neural development and wiring, but also in brain homeostasis. This study shows that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. These cells have been called MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. These findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. It is suggested that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before (Stratoulias, 2015b).

This study reports the identification of an unusual cell type, that has been called MiC, in the Drosophila brain. The appearance of MiCs was induced by three mechanisms: the induction of either immunity, or autophagy, or when the conserved neurotrophic factor DmMANF was downregulated, specifically in glia cells. It is concluded that in all three cases the cell type is the same because they are positive for the same markers, namely DmMANF, dSTAT, Zfh1, Relish and Draper, while they do not express Repo or Elav. MiCs were not observed when the same manipulations were done in neurons or in hemocytes or if they were limited only to subpopulations of glia. They were also not seen when using previously described Drosophila neurodegeneration models or various other manipulations (Stratoulias, 2015b).

The data suggest that MiCs produce an immune response. In MiCs, the JAK/STAT pathway is activated and the NF-κB factor Relish, which is the key activator of antibacterial peptide genes, is localized in the nuclei of MiCs. Consistent with this, it has recently been shown that the expression of constitutively active Relish in glia is sufficient to activate innate immune response and cause neurodegeneration in adult flies (Stratoulias, 2015b).

In addition, evidence is provided that MiCs are potentially phagocytic. First, MiCs express the engulfment receptor Draper, a protein that is essential and required for engulfment in a number of studies. Furthermore, MiCs have a very high lysosomal content, which suggests that they have a phagocytic function. On the other hand, no signs of endocytosis in MiCs, such as membrane internalizations or cellular debris, were identified. Consistent with these observations, recent data indicate that phagocytosis is not essential for microglia activation although they have phagocytic potential (Stratoulias, 2015b).

The data also point towards that MiCs are motile cells. First, they have elongated arms typical for migrating cells. Second, they have a random distribution in the brain appearing in neuropil areas which are known to be devoid of all cell bodies. Finally, dSTAT, a transcription factor which is known to specify and maintain cell motility, is localized in the nuclei of MiCs (Stratoulias, 2015b).

The functions described above, namely motility, production of pro-inflammatory mediators, expression of engulfment receptors , being positive for neurotrophic factors and more specifically the neurotrophic factor MANF are all features of macrophages/hemocytes and mammalian microglia. In addition, their appearance only in the CNS and the ventral nerve cord as well as their mode of emergence under brain homeostasis disturbance, resembles activation of mammalian microglia (Stratoulias, 2015b).

There is some evidence for the existence of microglia-like cells in other invertebrates such as leeches and mollusks. In cockroaches they have been reported to appear under in vitro conditions. However, microglia have not been identified in Drosophila. Rather, in flies glia are competent to perform immune-like functions such as engulfment of neuronal corpses during development and adulthood (Stratoulias, 2015b).

In vertebrates, microglia have been studied for more than 100 years. However, until recently their origin has been under controversy. Microglia, unlike glia and neurons, do not derive from the neuroectoderm. Instead they derive from macrophages produced by primitive hematopoiesis in the yolk sac. Similar to mammalian microglia, MiCs could also be of hematopoietic origin. In flies, macrophages/hemocytes, microglia or microglia-like populations have not been described in the CNS. MiCs did not appear when using hemocyte-specific Gal4 drivers either to knockdown DmMANF (and overexpress Dicer-2) or to induce immunity. Another possibility is that MiCs are circulating macrophages/hemocytes that infiltrate the brain upon genetically challenged conditions that may result in blood brain barrier disruption. Unfortunately, blood brain barrier disruption has not been studied during pupation and currently no experimental method exists for investigating blood brain barrier integrity. However, in situ hybridization data show that repo-Gal4; UAS-DmMANFRNAiUAS-Dicer-2 late pupal brains are not positive for the hemocyte marker Gcm, therefore MiCs cannot be (at least typical) hemocytes (Stratoulias, 2015b).

Alternatively, MiCs may originate from midline glia. As MiCs, midline glia do not express Repo. They are of mesoectodermal origin and have a distinct lineage from all other glial cells. During normal development, midline glia are eliminated by apoptosis in two temporally distinct waves, which results in midline glia not existing during late pupation and in adulthood. Interestingly, MiCs express the midline glia marker Single-minded. Therefore, it could be that MiCs indeed are midline glia that are not eliminated by apoptosis, but instead invade the neuropil areas. On the other hand, MiCs do not express the midline glia marker Slit, while they express Engrailed, a transcription factor that is not expressed in midline glia (Stratoulias, 2015b).

An exciting possibility is that MiCs may be either glia or neurons that under genetically challenged conditions transdifferentiate to MiCs and lose the expression of glial or neuronal markers. Very recently, a phagocytic cell type has been identified in Drosophila pupal brain. These cells are glia, they express Draper and they are Lysotracker positive. However, in contrast to MiCs these cells appear in the wild-type brain and are Repo positive. In addition they are localized specifically at the periphery of the neuropil and extend only their processes inside the neuropil. Finally, at the ultrastructural level (TEM) they do not show the characteristic MiC phenotype, namely multiple large lysosomes filled with transversely stacked membranes (Stratoulias, 2015b).

MiCs appear only transiently during metamorphosis, when a profound reorganization of the larval to adult CNS occurs. This cellular behavior may be vestigial from the evolution of Holometabola from hemimetabolous ancestors and it would be interesting to see if similar cells exist in normal conditions during brain development in species that undergo various forms of metamorphosis. It is proposed that MiCs differentiate from an earlier established cell population and do not divide or divide at very low rate during metamorphosis. Two lines of evidence support this assumption: first, MiCs do not stain with the mitotic marker PH3 and second, in the pupal brain the BrdU fed during larval stage is retained (Stratoulias, 2015b).

In summary, this study shows that by employing three different genetic mechanisms in vivo an unusual cell type appears in the Drosophila brain will be called MiC. MiCs express a unique set of molecular markers. These cells share many similarities with professional macrophages/hemocytes and vertebrate microglia. Macrophages/hemocytes, or microglia-like cells have not been previously identified in the Drosophila CNS. In addition, the pathways activated in MiCs, as well the molecular markers presented in this study, are evolutionarily well conserved from flies to humans, therefore making these results potentially relevant to higher organisms. Further investigations of MiCs’ origin, differentiation and stimuli that trigger them will help in gaining a better understand how immunity is attained in the CNS (Stratoulias, 2015b).

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons

In vertebrates the development and function of the nervous system is regulated by neurotrophic factors (NTFs). Despite extensive searches no neurotrophic factors have been found in invertebrates. However, cell ablation studies in Drosophila suggest trophic interaction between neurons and glia. This study reports the invertebrate neurotrophic factor in Drosophila, DmMANF, homologous to mammalian MANF and CDNF. DmMANF is expressed in glia and essential for maintenance of dopamine positive neurites and dopamine levels. The abolishment of both maternal and zygotic DmMANF leads to the degeneration of axonal bundles in the embryonic central nervous system and subsequent nonapoptotic cell death. The rescue experiments confirm DmMANF as a functional ortholog of the human MANF gene thus opening the window for comparative studies of this protein family with potential for the treatment of Parkinson's disease (Palgi, 2009).

This study has characterize the unique evolutionarily conserved NTF in invertebrates, DmMANF. Classically, the NTFs determine the number of neurons by supporting survival and antagonizing death. They also control neurite outgrowth and target innervation. CDNF and MANF support the survival of dopaminergic neurons in the rat models of neurotoxicity, preventing both neurite degeneration and neuronal death. However, whether these factors regulate the number of neurons during PCD is not known. DmMANF is clearly required in Drosophila for the maintenance of the DA neurites but not the neurites of serotonergic or the subpopulation of motoneurons. Surprisingly, despite the axonal degeneration in DmMANFΔ96 mutant larvae the somae of DA neurons persist. Moreover, some somae but not neurites of DA neurons persist even when their death was ectopically triggered by overexpression of the proapoptotic proteins. Thus, programmed death in the Drosophila DA neurons seems to follow a 'dying-back' pattern where the neurites degenerate first followed by the death of somae. Whether DmMANF is a bona fide NTF promoting the survival of DA neurons remains, however, open as the mutant larvae die before it can be judged. However, in the VNC of DmMANFΔ96mz mutants dying cells were observed with nonapoptotic ultrastructure. The exact identity of those cells remains undetermined but their location close to ventral midline suggests they are midline DA neurons dying after the loss of neurites. By TEM analysis, the elimination of DmMANF causes cell death resembling caspase-independent cell death, characterized by swelling of organelles, and the appearance of 'empty' spaces. All those characteristics including dilated and rounded ER, are observed in the DmMANFΔ96mz mutant VNC. Dilation of ER indicates ER stress and it has been recently shown that during ER stress MANF is upregulated. As DA neurons are highly susceptible to ER stress-induced cell death it could possibly explain why these neurons are specifically altered in DmMANFΔ96mz mutants. Also in mutants deficient of both maternal and zygotic DmMANF glia contain cellular debris indicating activation of glial engulfing activity. During metamorphosis glia accumulate highly electron-dense material when clearing axonal debris associated with neuronal remodeling. Taken together, it is concluded that DmMANF is the first invertebrate NTF required for maturation and maintenance of DA neurites from embryonic stage 16 onward at least to the second instar larval stage (Palgi, 2009).

The ability of HsMANF to replace the function of DmMANF suggests that these NTFs should share common signal transduction mechanisms including receptors. This makes the Drosophila model very attractive to study the MANF and CDNF signaling pathways by using the powerful fruit fly genetics. As human MANF and CDNF represent potential drug targets for the treatment of Parkinson's disease, the usage of well-established Drosophila disease models could be extremely important (Palgi, 2009).

Functions of Manf orthologs in other species

A comparative analysis of the molecular features of MANF and CDNF

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. This study focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to a previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) was investigated because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (DeltaCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or DeltaC CDNF, it was found that CDNF and DeltaCMANF hardly elevated the CRELD2 secretion. Effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion were investigated. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF (Norisada, 2016).

Mesencephalic astrocyte-derived neurotrophic factor is involved in inflammation by negatively regulating the NF-kappaB pathway

Inflammation can cause endoplasmic reticulum (ER) stress and therefore activates the unfolded protein response (UPR). ER stress and the consequent UPR have the potential to activate NF-kappaB. However, the factors mediating the crosstalk between ER stress and the NF-kappaB pathway remain unclear. This study determined that ER stress inducible protein Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) was up-regulated in autoimmune diseases and inflammatory disease models. Inflammation caused MANF to relocalize to the nuclei. MANF interacted with the DNA binding domain of p65 through its C-terminal SAP-like domain in the nuclei under the condition of inflammation or ER stress. MANF consequently inhibited p65-mediated transcriptional activation by interfering with the binding of p65 to its target genes promoters. Consistently, MANF suppressed the expressions of NF-kappaB-dependent target genes and the proliferation of inflammatory synoviocytes. These findings suggest that MANF may be a negative regulator of inflammation and mediate the crosstalk between the NF-kappaB pathway and ER stress (Chen, 2015).

MANF is indispensable for the proliferation and survival of pancreatic beta cells

All forms of diabetes mellitus (DM) are characterized by the loss of functional pancreatic beta cell mass, leading to insufficient insulin secretion. Thus, identification of novel approaches to protect and restore beta cells is essential for the development of DM therapies. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-inducible protein, but its physiological role in mammals has remained obscure. This study generated MANF-deficient mice that strikingly develop severe diabetes due to progressive postnatal reduction of beta cell mass, caused by decreased proliferation and increased apoptosis. Additionally, it was shown that lack of MANF in vivo in mouse leads to chronic unfolded protein response (UPR) activation in pancreatic islets. Importantly, MANF protein enhanced beta cell proliferation in vitro and overexpression of MANF in the pancreas of diabetic mice enhanced beta cell regeneration. It was demonstrated that MANF specifically promotes beta cell proliferation and survival, thereby constituting a therapeutic candidate for beta cell protection and regeneration (Lindahl, 2014).


Search PubMed for articles about Drosophila Manf

Airavaara M, Chiocco, M. J., Howard, D. B., Zuchowski, K. L., Peränen J., Liu, C., et al. (2010) Widespread cortical expression of MANF by AAV serotype 7: Localization and protection against a brain injury. Exp Neurol. 225: 104-113. PubMed ID: 20685313

Chen, L., Feng, L., Wang, X., Du, J., Chen, Y., Yang, W., Zhou, C., Cheng, L., Shen, Y., Fang, S., Li, J. and Shen, Y. (2015). Mesencephalic astrocyte-derived neurotrophic factor is involved in inflammation by negatively regulating the NF-kappaB pathway. Sci Rep 5: 8133. PubMed ID: 25640174

Glembotski, C. C., Thuerauf, D. J., Huang, C., Vekich, J. A., Gottlieb, R. A., Doroudgar, S. (2012). Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion. J Biol Chem. 287: 25893-25904. PubMed ID: 22637475

Lindholm, P., Voutilainen, M. H., Lauren, J., Peranen, J., Leppanen, V. M., Andressoo, J. O., Lindahl, M., Janhunen, S., Kalkkinen, N., Timmusk, T., Tuominen, R. K. and Saarma, M. (2007). Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature 448(7149): 73-77. PubMed ID: 17611540

Lindholm, P., Peränen, J., Andressoo, J-O., Kalkkinen, N., Kokaia, Z., Lindvall, O., et al. (2008). MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain. Mol Cell Neurosci. 39: 356-371. PubMed ID: 18718866

Lindahl, M., Danilova, T., Palm, E., Lindholm, P., Voikar, V., Hakonen, E., Ustinov, J., Andressoo, J. O., Harvey, B. K., Otonkoski, T., Rossi, J. and Saarma, M. (2014). MANF is indispensable for the proliferation and survival of pancreatic beta cells. Cell Rep 7(2): 366-375. PubMed ID: 24726366

Lindström, R., Lindholm, P., Kallijärvi, J., Yu, L-Y., Piepponen, T. P., Arumäe, U., et al. (2013). Characterization of the Structural and Functional Determinants of MANF/CDNF in Drosophila In Vivo Model. PLoS ONE 8(9):e73928. PubMed ID: 24019940

Lindström, R., Lindholm, P., Kallijärvi, J., Palgi, M., Saarma, M. and Heino, T. I. (2016). Exploring the conserved role of MANF in the unfolded protein response in Drosophila melanogaster. PLoS One 11: e0151550. PubMed ID: 26975047

Mizobuchi N, Hoseki J, Kubota H, Toyokuni S, Nozaki J-I, Naitoh M, et al. (2007). ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element. Cell Struct Funct. 32: 41-50. PubMed ID: 17507765

Neves, J., Zhu, J., Sousa-Victor, P., Konjikusic, M., Riley, R., Chew, S., Qi, Y., Jasper, H. and Lamba, D. A. (2016). Immune modulation by MANF promotes tissue repair and regenerative success in the retina. Science 353: aaf3646. PubMed ID: 27365452

Norisada, J., Hirata, Y., Amaya, F., Kiuchi, K. and Oh-hashi, K. (2016). A Comparative Analysis of the Molecular Features of MANF and CDNF. PLoS One 11(1): e0146923. PubMed ID: 26820513

Palgi, M., Lindström, R., Peränen, J., Piepponen, T. P., Saarma, M., Heino, T. I. (2009). Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons. Proc Natl Acad Sci U S A. 2009;106: 2429-2434. PubMed ID: 19164766

Palgi, M., Greco, D., Lindström, R., Auvinen, P., Heino, T. I. (2012). Gene expression analysis of Drosophila Manf mutants reveals perturbations in membrane traffic and major metabolic changes. BMC Genomics. 13:134. PubMed ID: 22494833

Stratoulias, V. and Heino, T. I. (2015a). Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain. Gene Expr Patterns 18: 8-15. PubMed ID: 25917377

Stratoulias, V. and Heino, T. I. (2015b). MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain. Cell Mol Life Sci 72(10): 1989-2004. PubMed ID: 25511196

Voutilainen, M. H., Back, S., Porsti, E., Toppinen, L., Lindgren, L., Lindholm, P., Peranen, J., Saarma, M. and Tuominen, R. K. (2009). Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease. J Neurosci 29(30): 9651-9659. PubMed ID: 19641128

Biological Overview

date revised: 10 April, 2017

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