Open Access

Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes

BMC Genomics201213:134

DOI: 10.1186/1471-2164-13-134

Received: 16 June 2011

Accepted: 11 April 2012

Published: 11 April 2012

Abstract

Background

MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf.

Results

The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well.

Conclusions

We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.

Background

Recently characterised MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) and CDNF (Cerebral Dopamine Neurotrophic Factor) form an independent family of neurotrophic factors [1]. MANF was identified from a conditioned media of cultured mesencephalic astrocytes in a search for secreted factors supporting dopamine (DA) neurons [2]. Specific loss of DA neurons is the characteristic feature of Parkinson's disease (PD). Therefore factors that maintain and support DA neurons are attractive candidates for the treatment of PD. MANF was shown to support the survival of cultured primary DA neurons but to have no effect on cultured GABAergic or serotonergic neurons [2]. Subsequently, mammalian MANF and its paralog CDNF were shown to prevent the loss of DA neurons in mouse 6-OHDA PD model [3, 4]. Contrary to other vertebrate neurotrophic factors the MANF/CDNF family is evolutionarily well conserved among multicellular organisms including the fruit fly, Drosophila melanogaster[2, 3, 5]. Importantly, the protective role of MANF for DA neurons is also conserved [5]. Apparently both mammals and invertebrates share the same signalling partners as the lack of Drosophila Manf can be substituted by human MANF [5]. However, the interaction partners or how these proteins act at the molecular level are still elusive. It is important to understand the mechanisms of how these MANF/CDNF family proteins work at molecular level before the potential therapeutic applications.

Recent studies have shown the protective role of mammalian MANF (also known as ARMET) to be more general than restricted to the nervous system. MANF is upregulated by UPR in several mammalian cell lines [68] and by ischemia induced UPR in the heart and brain [810]. ER is the central regulator of protein folding and quality control and it has to adapt its capacity to the specific need of a particular cell type. Conditions challenging the function of the ER, like an increase of newly synthesized unfolded proteins in its lumen, lead to UPR [11]. In eukaryotes, the three canonical branches of UPR are mediated by ER membrane-associated sensor proteins. In stress-free, functional ER the intralumenal domains of these sensor proteins are bound to a chaperone BiP/GRP78 (Binding immunoglobulin protein/Glucose-regulated protein 78) and maintained inactive [12, 13]. The UPR intersect with a variety of inflammatory and stress signalling pathways and networks activated by oxidative stress, all of which can influence cell metabolism. ER stress and UPR have also been implicated in the pathogenesis of several neurodegenerative diseases because of their characteristic accumulation of specific misfolded proteins [14]. Data from PD patients reveal that in DA neurons of substantia nigra the UPR is activated [15]. Recently, Drosophila has been used progressively to model human neurodegenerative diseases and UPR [1618].

We have previously generated and characterized Drosophila Manf mutants. The zygotic null mutants (Manf Δ96 ) survive up to 2nd instar larvae due to the high maternal contribution. Mutants lacking both maternal and zygotic Manf (Manf mzΔ96 ) are late embryonic lethal and never hatch [5]. The embryonic lack of maternal Manf gene products and the lethality is rescued by paternal Manf gene expression. Both Manf mzΔ96 and Manf Δ96 mutants share nervous system defects, particularly dopamine neurites are altered and degenerate. Ectopic overexpression of Manf reveals no evident abnormalities in the embryonic or larval nervous system development or in the adult flies (data not shown).

Here we compare the mRNA expression profiles of Manf mzΔ96 mutant embryos, Manf Δ96 mutant larvae, paternally rescued maternal mutant embryos Manf mΔ96 /+, and Manf ubiquitously overexpressing larvae to the wild type animals of exactly the same stages.

Results and discussion

The most prevalent changes in gene expression occur in Manf mutants that lack the maternal contribution of Manf

For microarray gene expression analysis we used two developmental stages in combination with three separate genotypes. The age of embryos and larvae were selected according to the lifespan of the Manf mutants. Manf mzΔ96 mutants fail in tracheal air filling and never hatch. Mutant Manf mzΔ96 embryos were picked during the late stage of 17 (21-22 hours after egg laying, AEL) just before hatching when the trachea of wild type embryos fill with air. Mutant Manf Δ96 larvae with maternal contribution survive to approximately 75 hours AEL and were collected as first instars 29-50 hours AEL when maternal loads of Manf gene products have decreased. Three biologically independent sets of samples were used for microarray analysis. The expression profiles of all transgenic and mutant animals were compared to the wild type of the corresponding developmental stage. The numerical overview of statistically significant differences (P < 0.01) showed the most prominent changes in gene expression of Manf mzΔ96 mutants (about 10% of genes differentially regulated). The smallest changes took place in the case of paternal rescue (less than 0.5% of genes differentially regulated) (Table 1). Among the differentially regulated genes, approximately half were up- or downregulated in different Manf conditions. Altogether we validated 40 genes of the microarray results. Genes were selected by several criteria such as differential expression or similar regulation in both mutants or otherwise high representation in the whole dataset. As a result, 61.5% of validated genes in different genetic conditions were independently confirmed by qPCR (Tables 2 and 3, Additional file 1: Table S1).
Table 1

Overview of microarray experiment

comparison between genotypes

diff. reg. probes

% all probes

down reg.

% all diff. reg.

upreg.

% all diff. reg.

Manf mz Δ 96 vs wt stage 17 embryos

3183

7.3

1501

47.2

1682

52.8

Manf mz Δ 96 / + vs wt stage 17 embryos

180

0.4

53

29.4

126

70.0

Manf mz Δ 96 vs Manf mz Δ 96 /+ stage 17 embryos

2681

6.2

1290

48.1

1391

51.9

Manf Δ 96 vs wt 29 -50 hr AEL larvae

1734

4.0

894

51.6

840

48.4

69B -GAL4> UAS-Manf 133 vs wt 29-50 hr AEL larvae

1240

2.8

513

41.4

727

58.6

Manf Δ96 vs 69B -GAL4 > UAS- Manf 133 29-50 hr AEL

2775

6.4

1615

58.2

1160

41.8

The number of differentially regulated probes was compared. In the Agilent Drosophila microarray design (4 × 44 K) there was unequal number of probes targeting the particular gene, ranging from one probe to several per gene.

Table 2

qPCR validation of results and microarray data obtained from stage 17 embryos

  

microarray

qPCR

  
  

Manf mz Δ 96 vs w

Manf mz Δ 96 vs w

69B> Manf 133 vs w

  

#

Gene Name

log FC

P.Value

log2

T-test

log2

T-test

gene ID

Description

1

CG10420

1.98

0.0001

5.15

9.7E-07

-3.29

1.8E-05

CG10420

Nucleotide exchange factor SIL1 precursor

2

CG14879

-4.03

7.3E-11

-3.46

0.0004

  

CG14879

Concanavalin A like lectin homology

3

CG5810

-1.92

1.8E-06

-3.78

1.5E-07

  

CG5810

leucine rich repeat

4

Ddc

1.62

4.6E-05

2.69

0.0003

-4.99

1.6E-05

CG10697

DOPA decarboxylase

5

DnaJ-H

1.10

4.1E-05

2.56

2.3E-05

  

CG9828

DnaJ homolog

6

Hrs

1.56

0.0001

1.38

0.05

  

CG2903

Hepatocyte growth factor-regulated tyrosine kinase substrate

7

Hsp83

2.46

9.7E-05

4.57

3.2E-06

  

CG1242

Heat shock protein 83, HSP90 homolog

8

InR

1.21

0.0007

1.87

3.4E-05

  

CG18402

Insulin-like receptor precursor

9

Pak3

-1.96

0.0003

-1.14

0.0003

  

CG14895

Pak, serine threonine kinase

10

Pi3K92E

1.11

0.0003

2.34

0.03

  

CG4141

phosphatidylinositol-4-phosphate 3-kinase

11

pipe

-1.53

4.2E-05

-0.25

0.0001

  

CG9614

heparan sulfate 2-O-sulfotransferase

12

pale

2.83

3.4E-07

4.83

1.0E-06

  

CG10118

TH; Tyrosine 3-hydroxylase

13

punch

1.97

4.3E-06

8.41

0.0001

0.69

0.02

CG9441

punch, GTP cyclohydrolase I

14

Rala

1.48

1.7E-07

0.63

2.1E-05

  

CG2849

Ras-related protein

15

sulfateless

4.21

1.8E-09

1.62

4.3E-05

  

CG8339

heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase

16

ROP

3.33

4.2E-10

0.82

4.3E-05

  

CG15811

Ras-opposite

17

ubisnap

-0.43

4.4E-05

-1.18

0.001

  

CG11173

ubisnap, SNAP29 homolog

18

DmManf

-1.37

0.0006

-5.39

6.3E-06

1.82

1.8E-06

CG7013

Manf, known previously as Arp-like

Only statistically significant results (P < 0.05) are presented. Results of qPCR were made comparable to microarray fold change (FC) by calculating the log2 from the relative fold changes. T-test means calculated P-value associated with Student's t-Test.

Table 3

qPCR validation of results and microarray data obtained from 29-50 hr larvae

  

microarray

qPCR

microarray

qPCR

 
  

Manf Δ 96 vs w

Manf Δ 96 vs w

Manf Δ 96 vs 69B> Manf 133

69B> Manf 133 vs w

69B> Manf 133 vs w

 

#

Gene Name

log FC

P.Value

log2

T-test

log FC

P.Value

log FC

P,Value

log2

T-test

gene ID

1

DmManf

  

-8.0

0.0001

-2.0

0.0002

  

3.2

6.0E-07

CG7013

2

CG14879

-3.2

5.5E-06

-11.5

0.0002

-2.7

4.4E-05

  

-3.9

1.5E-06

CG14879

3

cycle

-0.5

0.0002

-7.2

0.0001

-1.0

4.7E-09

0.5

3.2E-05

-5.0

9.0E-08

CG8727

4

nol

-1.1

0.0003

-4.3

1.7E-05

-1.9

3.9E-07

1.1

6.3E-06

0.8

0.0005

CG32077

5

Pak3

-2.8

3.1E-07

-5.8

5.2E-05

-2.7

3.8E-07

    

CG14895

6

pipe

-0.9

0.0003

-1.2

3.7E-06

-1.0

1.8E-05

0.9

4.1E-05

  

CG9614

7

Rala

-1.4

2.4E-08

-2.0

0.0001

1.4

5.7E-09

0.9

4.2E-06

  

CG2849

8

Rep

-2.0

0.0001

-3.3

4.6E-07

      

CG8432

9

sip3

-0.5

0.0003

-9.6

6.4E-06

      

CG1937

10

Sk2

-0.6

0.0007

-8.7

1.2E-05

-1.6

0.0003

0.6

0.0003

-12.2

4.5E-08

CG32484

11

Ubc-E2H

0.7

0.0001

1.2

9.9E-05

      

CG2257

12

Pi3K

-1.0

5.5E-05

-9.9

0.0003

  

-1.0

8.0E-05

  

TC208938

13

Uch-L3

-0.6

0.0003

-0.4

0.0001

-0.4

0.0004

    

CG3431

Only statistically significant (P < 0.01) results are presented. Results of qPCR results were made comparable to microarray fold change (FC) by calculating the log2 of the relative fold changes. T-test means calculated P-value associated with Student's T-test.

Membrane transporters and metabolic genes are downregulated in Manf mzΔ96 mutants

Development of maternal and zygotic mutant Manf mzΔ96 proceeds until stage 16 with no differences to wild type embryos, but 21 hours AEL the cuticle and the nervous system defects become evident [5]. In comparison to wild type embryos of the same age, in Manf mzΔ96 mutants 1191 genes were found to be downregulated. These genes were grouped into 105 functional clusters (Additional file 2), among which the most significantly enriched clusters were related to membrane transporters (25 genes) and transmembrane proteins (146 genes) (Table 4). There were several enriched clusters referring to different metabolic processes such as amine, amino acid and carboxylic acid catabolic processes (11 genes), DNA metabolic processes (26 genes), and genes related to pyrimidine metabolism (15 genes).
Table 4

GO clustering analysis of downregulated genes in Manf mzΔ96 mutants

 

GO term

enrichment score

gene number

1

membrane transporters

2.9

25

2

transmembrane proteins

2.3

146

3

amine catabolic processes

2.1

11

4

mitochondrion

2.0

28

...

DNA replication, DNA metabolic process

1.7

26

...

pyrimidine metabolism

1.5

15

...

ncRNA, rRNA metabolic process, ribosome biogenesis

1.4

23

...

structural constituent of chitin-based cuticle

1.4

14

...

Hox, DNA dependent transcription regulation

1.3

122

...

organophosphate, glycerolipid metabolic process

1.3

18

...

neurological system process

1.0

44

Clusters are represented starting from the highest enrichment score in diminishing order. Only the highest clusters are shown, for the full list of DAVID cluster analysis of downregulated genes in Manf mzΔ96 mutants, see Additional file 2. In the case of missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.

Among the downregulated genes in Manf mzΔ96 mutants, the gene ontology (GO) term of mitochondria-related transcripts was highly enriched (28 genes). Mitochondria are the respiratory machinery of the cell responsible for oxidation processes and participate in maintaining cellular homeostasis. The lack of Manf causes downregulation of several components in all mitochondrial compartments: the lumen as well as the inner and outer membranes.

Stress and defence response related genes are induced in Manf mzΔ96 mutants

In Manf mzΔ96 mutant embryos, 1243 genes were upregulated in comparison to wild type embryos of the same stage (Additional file 3). The most prominent group of significantly enriched GO terms was immune and defense response (69 genes), followed by the groups related to proteolysis, hydrolases and peptidases (197 genes) (Table 5). The upregulated gene set was also enriched in genes related to actin cytoskeleton organization and actin filament-based process (28 genes). Moreover, genes involved in cell death (28 genes) were prominently enriched as well.
Table 5

GO clustering analysis of upregulated genes in Manf mzΔ96 mutants

 

GO term

enrichment score

gene number

1

defense response, immune response

9.4

69

2

endopeptidase activity, proteolysis

6.4

197

...

peptidase inhibitor and enzyme inhibitor activity

4.0

24

...

stress response, response to abiotic stimulus

3.4

31

...

plasma membrane part, integral to plasma membrane

3.3

59

...

actin cytoskeleton organization

3.1

28

...

extracellular region part

2.8

30

...

cell death

2.7

28

...

embryonic and epithelial morphogenesis, cell polarity

2.5

67

...

cell adhesion

1.7

25

...

membrane invagination, phagocytosis, vesicle-mediated transport

1.6

50

...

apical junction, cell-cell junction assembly and organisation

1.6

22

Clusters are represented starting from the highest enrichment score in diminishing order. Only the highest clusters are shown, for the full list of DAVID cluster analysis of upregulated genes in Manf mzΔ96 mutants, see Additional file 3. In the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.

Enzymes for dopamine synthesis are upregulated despite of low dopamine levels

Extremely low dopamine levels are detected in Manf mzΔ96 embryos [5]. Nonetheless, we observed significant upregulation of transcripts of the dopamine producing enzymes tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc) (Table 2). Punch; encoding a GTP cyclohydrolase, an enzyme for tetrahydrobiopterin (a cofactor for TH) synthesis was also upregulated in Manf mzΔ96 embryos (Table 2). There could be several explanations for these alterations. Tyrosine, the essential amino acid for dopamine synthesis, is transported into the cell from hemolymph. In Manf mutants several amino acid membrane transporters were downregulated. The lack of substrate, tyrosine, together with low amounts of the end product, dopamine, could lead to the upregulation of the genes coding for the enzymes in dopamine synthesis pathway and their cofactors.

Genes involved in nucleic acid metabolism and protein folding are downregulated in larval Manf Δ96 mutants

Larval Manf Δ96 mutants with maternally contributed Manf gene products never reach 3rd instar stage and rarely survive up to 75 hours AEL at 25°C. Initially, Manf Δ96 mutant larvae hatch and feed normally. Whereas wild type larvae grow rapidly, the mutant larvae remain smaller and start to wander away from food, become sluggish and stop moving, still responding to touch and usually die during the first larval molt [5]. Because of the temporal differences in survival between individual Manf Δ96 mutant larvae from 1st to 2nd instar, for microarray analysis we collected larvae 29-50 hours AEL.

When comparing the expression profile of larval Manf Δ96 mutants to the wild type larvae, almost half the number of genes (690) was significantly downregulated as compared to the rate in Manf mzΔ96 embryos resulting in 140 functional clusters (Additional file 4). The most enriched GO terms fell into clusters related to intracellular organelle lumen and nucleic acid metabolic processes (Table 6). The cellular activities such as DNA replication, RNA processing and splicing were enriched among downregulated genes. The 5th highly enriched cluster consisted of GO terms such as ER related genes (24 genes), proline and arginine metabolism (9 genes), and oxioreductases (9 genes). Mitotic cell cycle, chromosomal segregation, and mitotic spindle organization were also clustered as significantly enriched. These changes could be linked to UPR, as one of the outcomes of UPR is general and unspecific downregulation of novel protein synthesis, at the same time activating the protein synthesis for chaperones and genes enhancing the protein folding to release the unfolded protein load in ER.
Table 6

GO clustering analysis of downregulated genes in Manf Δ96 larval mutants

 

GO term

enrichment score

gene number

1

nuclear lumen, intracellular organelle lumen

3.9

50

2

ncRNA, rRNA metabolic process

3.7

23

3

chromosome, non-membrane-bounded organelle

3.7

72

4

DNA replication, DNA metabolic process

3.4

29

5

prolyl 4-hydroxylase, oxioreductase, ER part

3.4

24

6

RNA, mRNA metabolic process, spliceosome

2.8

45

...

mRNA transport, nuclear transport, nuclear export

1.6

14

...

chromosome condensation, DNA packaging

1.6

15

...

cell cycle, mitosis, chromosome segregation

1.3

59

Clusters are represented starting from the highest enrichment score in diminishing order. Only the highest clusters are shown, for the full list of DAVID cluster analysis of downregulated genes in Manf Δ96 mutants, see Additional file 4. In the case on missing order number replaced by "..." there were several clusters higher in order describing similar processes that have been listed already above.

Sugar metabolism, hydrolases, and ER related oxidation reduction genes are induced in Manf Δ96 larvae

In Manf Δ96 larval mutants, 682 genes showed upregulation in comparison to the wild type larvae. The most enriched functional clusters included GO terms like sugar metabolism and glucosidases, glycosyl hydrolases (18 genes), and hydrolases and carboxylesterases (23 genes), followed by cluster of monooxygenases, Cytochrome P450, iron, vesicular fraction, oxidation reduction and endoplasmic reticulum (49 genes) (Additional file 5). Chitin and polysaccharide metabolism was also among the highly enriched GO terms (40 genes). The 5th ranked cluster of GO terms was immune and defence response (19 genes), which was the most highly enriched cluster in Manf mzΔ96 mutant embryos.

Genes related to RNA metabolism, ATP binding, and DNA replication are downregulated in both Manf mutants

Next, we looked for functional terms among the 208 commonly downregulated genes in both Manf mutants (Additional file 6). There was 30% of overlap in gene sets between the Manf Δ96 and Manf mzΔ96 mutants. Among the downregulated overlapping genes, the enrichment of GO terms fell into RNA metabolism and ribosome biogenesis (19 genes). Around 10% of all known ATP binding genes were downregulated (28 genes) together with 14 genes of the purine and pyrimidine metabolism. Additionally, the genes coding sugar transporters and the genes involved in transmembrane transport (7 genes) highly represented among downregulated genes in Manf mzΔ96 mutant embryos, were repressed in zygotic mutant Manf Δ96 as well. GO terms related to DNA replication (15 genes) were also enriched among the commonly downregulated genes in both Manf mutants.

Altogether, the Manf mzΔ96 mutant embryos lacking both maternal and zygotic Manf showed twice more drastic decline from wild type transcriptome than Manf Δ96 larval mutants, whose maternal transcripts gradually diminish. Beside behavioural and growth phenotype, Manf Δ96 larvae show degeneration of dopaminergic neurites in ventral nerve cord [5]. We found three genes downregulated in both mutants that are involved in neurite development: Abelson tyrosine kinase (Abl), Guanine nucleotide exchange factor GEF64C (Gef64C) and the transcription factor longitudinals lacking (lola).

A third of all upregulated genes (229) were induced in both mutants (Additional file 7). Immune and defense response was the most enriched functional cluster (29 genes) along with the group consisting of monooxygenases, oxidoreductases, vesicular fraction, endoplasmic reticulum, Cytochrome P450 and lipid metabolic process (21 genes). Controversially, disabled (Dab, substrate of Abl), was upregulated among the 10 genes involved in neuronal development e.g. transcription factor Krüppel (Kr), negative regulator of growth shrub (shrb), insulin receptor (InR), and Drosophila extracellular-signal-regulated kinase (ERK) rolled (rl).

Genes related to UPR were upregulated in Manf mutants

Previous in vitro studies using tunicamycin, the inhibitor of glycosylation, to induce ER stress in mammalian cell lines have shown in UPR the upregulation of MANF [6, 7]. In rat neonatal cardiomyocytes in response to UPR MANF is secreted to promote cellular survival [8]. ER stress and one of the consequences, UPR, has been mainly studied in yeast and mammalian cells. In Drosophila, there are several recent studies where UPR has been addressed [16, 19]. Manf has been shown to be upregulated after feeding tunicamycin to adult fruit flies indicating the involvement of Manf in chemically induced UPR in Drosophila[20].

To find out the intracellular localisation of Manf in Drosophila, we used larval 2nd instar garland cells. Garland cells are nephrocytes with high rate of endocytosis and express several neuronal and exocytosis markers e.g. prospero (pros, mammalian Prox-1 homolog), SNARE binding protein Ras opposite (Rop) facilitating neurotransmitter secretion, and Syntaxin 1A (Syx1A, a t-SNARE) [21, 22]. These cells have the most abundant expression of Manf starting from embryogenesis [5]. In the garland cells, Manf was localised around the nucleus, partially overlapping with ER-targeted marker (Figure 1A-C).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-134/MediaObjects/12864_2011_Article_4000_Fig1_HTML.jpg
Figure 1

Manf localises intracellularly partially to ER and endosomal compartment. A-C - The confocal micrographs of 2nd instar larval garland cells stained for α-Manf (magenta) showing Manf expression around the nuclei (A) overlapping partially with ER-EYFP marker (green), DAPI (blue) was used to stain nuclei (A-C). D - Western blot analysis shows two fold increased amount of phosphorylated elF2α in Manf mzΔ96 embryos in comparison to wild type w 1118 embryos. Decreasing amounts of samples were loaded to obtain the optimal result for quantification; the triangles represent the direction of decrease in loading. α-tubulin (α-tub) was used as a loading control. E-G - The confocal micrographs of Schneider-2 cells transfected with Manf cDNA construct and stained with Lysotracker (green) and α-DmManf show almost no colocalisation (less than 0.3%). H-M - The confocal micrographs of the wild type 3rd instar larval fat body expressing GFP-tagged UAS-constructs (green) driven by fat body specific ppl-GAL4 and stained for α-DmManf (magenta); nuclear stain DAPI (blue) was used. In H-J Manf localises close to clathrin coated vesicles marker GFP-clathrin light chain (Clc). In K-M Manf shows partial colocalisation with late endosomal compartment marker Rab7. N-S - In the salivary gland cells of 3rd instar larvae Manf (magenta) localises close to the basal cell borders and colocalises partially with early endosomal marker Rab5 (green) (N-P) and the recycling endosomal pathway marker Rab11 (green) (Q-S). Close arrows mark the cell borders and the open arrows mark the areas of colocalisation; all images consist of single laser confocal section. Scale bars: in A-C 2 μm, 4 μm in H-J, 5 μm in E-G and K-S.

Next, we tested the hypothesis that the metabolic changes in Manf mutant could be the result of severe ER stress caused by altered expression of ER related genes. Drosophila genes homologous to several ER stress pathway have been identified. Out of 30 genes involved in ER and protein processing in the KEGG database, 24 have one or more homologues in fruit flies (Figure 2, Table 7). Of these UPR related Drosophila genes, 30% showed altered gene expression in our microarray experiment. Altogether, 29 genes involved in ER and protein processing show statistically significant expression changes. The gene CG10420 is an annotated gene with unknown function in Drosophila. Its human homologue nucleotide exchange factor SIL1 (S. cerevisiae ER chaperone homologue) is a BiP binding protein. In humans, several mutations in SIL1 gene disrupting the protein cause the Marinesco-Sjögren syndrome (MSS), an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy [23]. We validated CG10420 by qPCR as downregulated by Manf overexpression and upregulated when Manf is abolished in Drosophila embryos and larvae. It has been shown by immunoprecipitation studies that mammalian MANF binds to BiP [24]. Thus it is possible that Manf and CG10420 compete in binding to BiP together with unfolded proteins. As the ectopic overexpression of Manf has no effect on fruit fly viability or nervous system development (data not shown), the diminished transcript level for CG10420 is not comparable to the total lack of this gene product in the MSS patients. According to our qPCR validated microarray results several other genes implicated in UPR were downregulated in larvae overexpressing Manf, such as pancreatic eIF-2α kinase (PERK), Heat shock protein 83 (Hsp83), Ubiquilin (Ubqn), and septin interacting protein 3 (sip3). In embryonic Manf mzΔ96 mutants all above mentioned genes were significantly upregulated as well as considerable number of other ER chaperone genes (Table 7). Furthermore, when evaluating the ultrastructural changes in Manf mzΔ96 mutants, we noticed that the ER was swollen and dilated in epidermal cells, indicating severe disturbances of ER structure (Figure 3A-C). In Manf mzΔ96 mutant embryos the extent of phosphorylated eukaryotic initiation factor eIF2α was more than two fold upregulated when compared to the wild type (Figure 1D) indicating the presence of UPR in these Manf mutants. The phosphorylation of eIF2α by PERK is a hallmark for UPR, resulting in reversible blockage of translation and downregulation of the protein load to the ER [25]. In Drosophila there are two kinases, PERK and Gcn2, shown to be able to phosphorylate eIF2α [26, 27]. The expression of Gcn2 is high only during early stages of embryogenesis [27]. Thus PERK is a potential candidate kinase behind eIF2α phosphorylation at the end of embryogenesis. Interestingly, our microarray data showed that in Manf mzΔ96 mutants the transcription of PERK was [25] upregulated and the genes involved in different metabolic processes such as amino acid, DNA and pyrimidine metabolism were downregulated indicating overall inhibition of translation. So it is probable that the UPR PERK pathway is activated in Manf mzΔ96 mutants. The second UPR sensor, IRE1, activates two separate downstream branches. One of the branches leads to the activation of Jun kinase and death pathway [28]. According to the microarray results, Drosophila Jun kinase kinase hemipterous showed significant upregulation in both Manf mutants.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-134/MediaObjects/12864_2011_Article_4000_Fig2_HTML.jpg
Figure 2

Protein processing in endoplasmic reticulum is altered in Manf mutants. An online coloured KEGG pathway scheme showing altered gene expression in red (upregulation), blue (downregulation), or purple (altered gene expression, differences between the two mutants) boxes. The unaltered known Drosophila homologues to identified components from other organisms are presented in green-filled boxes. The complete list of altered genes is summarised in Table 7. Notice the upregulation of genes encoding BiP/GRP78 chaperone binding proteins and components in ER leading to terminally misfolded protein degradation pathway. Out of the three branches of ER stress, IRE1 pathway leading to cell death shows upregulation in both mutants as PERK pathway is upregulated in maternal and zygotic Manf mutants.

Table 7

List of genes with altered expression according to microarray analysis related to KEGG pathway of protein processing in ER

KEGG

 

embryo

larvae

 

name

gene

o.ex.

mut.

o.ex.

mut.

description

NEF

CG10420

down

up

down

up

SIL1, BiP-associated protein, mutated in MSS

Hsp90

hsp83

 

up

down

down

Heat shock protein 83

PERK

pek

 

up

down

down

PEK; pancreatic eIF-2alpha kinase, UPR sensor

Hrd1

sip3

 

up

down

down

septin interacting protein 3, ERAD

DSK2

ubiqn

 

up

down

down

ubiquilin, ubiquitin-ass./transl. elongation factor EF1B

Cul1

lin19

 

down

up

up

lin-19-like, ubiquitin protein ligase, cullin homology

Skp1

skpC

 

down

up

down

skpC, E3 ubiquitin ligase

EDEM

Edem1

  

up

down

Edem1, Glycoside hydrolase

Ubx

p47

  

down

down

p47, human NSFL1

Skp1

skpE

 

down

 

down

RNA polymerase II transcription elongation factor

Sec23/24

CG1472

down

  

down

COP complex II, mediator of selective export from ER

UGGT

CG6850

   

down

UDP-glucose-glycoprotein glucosyltransferase

DOA10

CG1317

 

down

  

E3 ubiquitin-protein ligase MARCH6

Sec13/31

CG6773

 

up

up

up

involved in export from ER, nuclear import, cuticle development

MKK7

CG4353

 

up

 

up

hemipterous, Jun kinase kinase

Hsp70

Hspc1

   

up

Heat shock protein cognate 1

Ubc6/7

CG5823

   

up

ubiquitin-conjugating enzyme E2 J2

Hsp40

dnajh

 

up

  

DnaJ homolog subfamily B member 11

NEF

CG10973

 

up

  

hsp70-interacting protein

Bap31

CG13887

 

up

  

B-cell receptor-associated protein 31

NEF

CG2918

 

up

  

hypoxia up-regulated 1

sHSF

l(2)efl

 

up

  

lethal (2) essential for life

IRE-1

ire-1

 

up

  

inositol requiring enzyme 1, Ser/Thr kinase, UPR sensor

Hsp70

Hsp68

 

up

  

Heat shock protein 68

UbcH5

Ubce2

 

up

  

Ubiquitin conjugating enzyme 2

Hsp70

Hspc2

 

up

  

Heat shock protein cognate 2

NEF

CG7945

 

up

  

BCL2-associated athanogene 2

Sec13/31

CG8266

 

up

  

COP II complex, secretory vesicle budding from ER

S2P

S2P

 

up

  

endopeptidase

Significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. Gene name stands for the particular homologue gene name in Drosophila. Notice that the same KEGG identifier can lead to several different genes. p.res = paternal rescue, mut = mutant, o.ex. = overexpression.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-134/MediaObjects/12864_2011_Article_4000_Fig3_HTML.jpg
Figure 3

Manf mutants show severe defects in vesicular traffic in the cells with high secretion or endocytosis rate. A-C - In the epidermal cells of stage 17 Manf mzΔ96 mutant embryos compared to wild type (w), ER is rounded and swollen (open arrow), there are multiple vesicles stuck close to plasma membrane (thick arrow), and the cuticle (cu) is severely disorganized. Cuticular layers are indicated by a line. nu = nucleus D-E - High pressure freezing TEM images of 1st-2nd instar larvae show no difference in the layering of the cuticle between wild type and Manf Δ96 mutant. The cellular membranes are weakly stained by this technique. Notice the unattached first instar cuticle in Manf Δ96 mutant (cu' in E). F-G The comparison of wild type and Manf Δ96 mutant garland cells shows excessive accumulation and enlargement of α-vesicles (α) and dilated ER. The labyrinth channels and slit membranes are similar between wild type and Manf mzΔ96 mutant (white open arrows). H-I Secretory cells of gastric caeca in Manf mzΔ96 mutant show accumulation of vesicles full of debris (white arrow heads) never found in wild type. Scale bars: in A-E 500 nm, 1 μm in F and G, 2 μm in H and I.

Conclusively, the absolute lack of Manf results in severe ER stress and upregulation of many genes involved in UPR finally leading to the cell death. When maternal Manf stores are gradually decreased in Manf Δ96 larvae, there are only few genes upregulated that are related to ER: CG10420, ubiquitin protein ligase lin19, heat shock protein cognate 1 and ubiquitin-conjugating enzyme CG5823. As our data come mostly from gene expression analyses, further biochemical experiments are needed to identify the exact role of Manf in UPR.

Lack of Manf results in downregulation of several genes in exocytosis pathway

Ultrastructural study of Manf mzΔ96 mutants revealed overload of vesicles next to the apical part of epidermal cells and reduced microvillae thought to enhance the capacity of the secretion of these cells (Figure 3C). This result, together with the severe defects observed in the cuticle secretion and organisation (Figure 3B, C), suggested a possible involvement of the genes of the exocytosis pathway. Indeed, the expression of several genes related to exocytosis and SNARE transport were altered in different Manf conditions (Table 8 Additional file 8). In Manf mutants, several genes implied in exocytosis and vesicle transport from Golgi complex to the plasma membrane were downregulated (Syx1A, Syx6, SNAP29), whereas the ER residing syntaxins - Stx17 and Stx18 - were upregulated. This supports an inhibition of secretion from Golgi complex to the plasma membrane as seen in Manf mzΔ96 mutant epidermal cells in vesicle accumulation close to the apical area (Figure 3C).
Table 8

List of genes with altered expression according to microarray analysis related to KEGG pathway of exocytosis and SNARE complexes

KEGG

 

embryo

larvae

 

name

gene

o.ex.

mut.

o.ex.

mut.

description

Bos

CG4780

up

up

up

down

membrin

Vti1

koko

  

up

down

kokopelli; cyclin-dependent protein kinase regulator

SNAP29

usnp

up

down

 

down

usnp; ubisnap

Stx6

Syx 6

up

down

  

Syntaxin 6

 

Syt7

 

down

down

down

calcium-dependent phospholipid binding, Synaptotagmin.

Stx1-4

Syx 1A

  

down

down

Syntaxin 1A

Stx17

Syx 17

  

down

up

synaptic vesicle docking; neurotransmitter secretion

 

Synd

 

up

 

up

neurotransmitter secretion; synaptic vesicle endocytosis

Stx13

Syx 13

 

up

  

SNAP receptor: cytokinesis after mitosis and meiosis

 

Syb

up

up

  

SNAP receptor: synaptic vesicle docking in exocytosis

Syx18

Syx 18

up

up

  

Syntaxin 18

Bet1

CG14084

up

up

  

Bet1

VAMP7

CG1599

up

up

  

vesicle-associated membrane protein 7

Significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. Gene name stands for the particular homologue gene name in Drosophila. Few related Drosophila genes associated with neurotransmission missing from KEGG pathway were added. The according coloured scheme for exocytosis and SNARE of KEGG pathway is presented in Additional File 8. p.res = paternal rescue, mut = mutant, o.ex. = overexpression.

Expression of genes involved in cuticle development were altered in Manf mzΔ96 mutants

We have previously shown that Manf mzΔ96 embryos have disorganized cuticle [5]. At the end of embryogenesis from stage 16 onward, the cuticle components are secreted by epithelial cells and stored in regular layers, and subsequently the cuticular proteins are crosslinked by dopamine-derived quinones [29, 30]. Among the downregulated genes in Manf mzΔ96 embryos, there were 14 genes coding the structural components of the insect cuticle. At the same time, several other genes responsible for cuticle development were upregulated, such as the genes encoding enzymes involved in chitin synthesis, krotzkopf verkehrt (kkv, chitin synthase-1) [31, 32], knickkopf (knk, N-glycosylated membrane-bound extracellular protein involved in chitin microfibril formation) [33], and Syx1A (responsible for cuticle component secretion). Additionally, several genes involved in epithelial development and morphogenesis were upregulated and significantly enriched among the GO terms (35 genes) (Table 5 and Additional file 3).

We used transmission electron microscopy (TEM) analysis in Manf mzΔ96 mutants at the embryonic stage 17 to investigate the epithelial cells responsible for cuticle secretion. Indeed, these cells showed morphologically abnormal ER and accumulation of vesicles in the apical part (Figure 3A-C). It is possible that the enhanced endocytosis and disturbed exocytosis, together with misbalance in cuticular components, lead to disorganised and disrupted cuticle in Manf mzΔ96 mutant embryos. In larval Manf Δ96 mutant with gradually fading maternal contribution, the cuticle showed no disruption and the chitin layers were deposited and organised normally (Figure 3D, E). Instead there were problems in shedding the old cuticle and often the 1st instar cuticle remained attached (Figure 3E). This implies that the maternal loading of Manf gene products in larval Manf Δ96 mutants was sufficient to overcome defects in early cuticle development, secretion and layering, but insufficient to complete the first molt.

Large vesicles filled with electron dense debris are accumulated in Manf mzΔ96 mutant

To investigate the routes of membrane trafficking we evaluated genes involved in endocytosis. Of all Drosophila homologues known to be involved in endocytosis, 47% showed significant expression changes in our microarray experiment (Figure 4, Table 9). Genes coding for components of multivesicular body formation were especially altered. Several transmembrane receptors of growth factors were downregulated in Manf mutants and upregulated when Manf was overexpressed. PDGF- and VEGF-receptor related Pvr was upregulated in larvae in both lack and overexpression of Manf. Cbl, an E3 ubiquitin ligase and negative regulator of tyrosine kinase receptor signalling, was downregulated in mutant larvae and upregulated under Manf overexpression conditions. Two different members of endosomal recycling pathway, PAR family members and Rab-protein 11 (Rab11) were upregulated in mutants. PAR transcripts were upregulated by Manf overexpression as well.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-134/MediaObjects/12864_2011_Article_4000_Fig4_HTML.jpg
Figure 4

Changes in gene expression related to endocytosis in Manf mutants. An online coloured KEGG pathway showing altered gene expression in red (upregulation), blue (downregulation), or purple (altered gene expression, differences between the two mutants) boxes. The unaltered components of the pathway with Drosophila homologues are coloured in green. White boxes represent the pathway members identified from other organisms with an unknown Drosophila homologue. The complete list of altered genes is summarised in Table 9. Notice the abundant expression changes in genes encoding proteins localised close to the plasma membrane and receptor tyrosine kinase internalisation pathway components. The changes of expression were highest among genes which products localise to multivesicular body and late endosomal compartments. Two branches of recycling pathway show upregulation of gene expression of key components PAR3, PAR6, and Rab11.

Table 9

List of genes with altered expression according to microarray analysis related to endocytosis KEGG pathway

KEGG

Gene

embryo

larvae

 

name

name

o.ex.

mut.

o.ex.

mut.

description

RTK

htl

   

down

Fgf receptor

RTK

EGFR

  

up

down

Egf receptor

ArfGAP

GAP69C

 

down

up

down

GTPase-activating protein 69C

VPS25

VPS25

   

down

Vacuolar protein sorting 25

beta-arrestin

krz

  

up

down

kurtz, β-arrestin

E3 ligase

Traf6

up

down

 

down

TNF-receptor-associated factor 6

E3 ligase

elgi

up

down

  

early girl

GRK

GPCRK 1

up

down

  

G protein-coupled receptor kinase 1

RTK

Ret

up

down

  

Ret oncogene

Cbl; E3 ligase

Cbl

up

up

up

down

ubiquitin mediated proteolysis

VPS37

CG11155

up

down

down

 

ESCRT-I complex subunit VPS37

PIP5K

CG17471

up

down

down

up

1-phosphatidylinositol-4-phosphate 5-kinase

CHMP4

shrb

down

up

down

up

shrub, charged multivesicular protein

Hsc70

Hspc 2

 

up

down

up

Heat shock protein cognate 2

FIP

nuf

down

up

down

up

nuclear fallout, Rab11 interacting protein

Epsin

lfs

 

up

down

up

liquid facets

endophilin

endoB

  

down

up

endophilin B

CHMP6

Vps20

down

up

  

vacuolar protein sorting 20

Clathrin

Chc

down

up

  

Clathrin heavy chain

TSG101

TSG101

down

up

  

tumor suppressor protein 101

STAM

Stam

   

up

signal transducing adaptor molecule

VPS28

Vps28

 

up

 

up

vacuolar protein sorting 28

PAR6

par-6

 

up

up

up

partitioning defective 6

Hsc70

Hspc 1

  

up

up

Heat shock protein cognate 1

Hsc70

Hsp 68

up

up

  

Heat shock protein 68

RTK

Pvr

up

up

  

PDGF- and VEGF-receptor related

ARFGTPase

CG7435

up

up

  

ADP ribosylation factor 84F

ArfGAP

CG30372

up

up

  

SH3 domain, ANK repeat and PH domain

ArfGAP

cenB1A

up

up

  

centaurin beta 1A

VPS4

Vps4

up

up

  

vacuolar protein sorting 4

CHMP2

CG14542

up

up

  

charged multivesicular body protein 2A

CHMP2

CG4618

up

up

  

charged multivesicular body protein 2B

MVB12

CG7192

up

up

  

ESCRT-I complex subunit MVB12

Hrs

Hrs

up

up

  

HGF regulated tyrosine kinase substrate

PAR3

baz

up

up

  

bazooka, partitioning defective 3

VTA1

CG7967

up

up

  

vacuolar protein sorting-associated protein

CHMP5

CG6259

 

up

  

charged multivesicular body protein 5

CHMP1

CG4108

 

up

  

charged multivesicular body protein 1

ArfGAP

cenG1A

 

up

  

centaurin gamma 1A

Rab11

Rab 11

 

up

  

Rab-protein 11

Significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. Gene name stands for the particular homologue gene name in Drosophila. Notice that the same KEGG identifier can lead to several different genes in Drosophila. The according coloured scheme for endocytosis KEGG pathway is presented in Figure 4.

To visualise Manf expression at subcellular level we used 3rd instar larval salivary gland cells that are the largest ones found in Drosophila. In the basal part, there was partial colocalisation of Manf expression with GFP-Rab11 (Figure 1Q-S) as well as with early endosomal marker GFP-Rab5 (Figure 1N-P). In larval fat body large cells with high secretory capacity GFP-clathrin light chain (Clc), a marker for clathrin coated vesicles, colocalised with Manf in some structures (Figure 1H-J). Manf localised close to GFP-Rab7, an important player in trafficking between the early and late endosomes and lysosomes, showing weak colocalisation (Figure 1L-N). Thus Manf localises to the endosomal structures with markers Clc, Rab5, Rab7, and Rab11; but probably does not share the same protein complexes with them.

Ultrastructural analysis of Manf mzΔ96 mutant stage 17 embryos revealed that the cells of secretory tissues such as gastric caeca, contain huge vesicles filled with cellular debris resembling multivesicular bodies and autophagosomes (Figure 3I). These structures were clearly missing in wild type embryos of the same age (Figure 3H). It is possible that these vesicles contain the misfolded proteins to be degraded or, alternatively, that the autophagy pathway is activated. The accumulation of vesicles full of debris to be degraded could be also due to the blockage in endosomal trafficking or lysosomal degradation.

Lysosomal genes are downregulated in Manf mutants

Because we detected in secretory cells the accumulation of multivesicular body like structures, is it possible that the lysosomal digestion mechanism was altered. Our microarray analysis revealed transcriptional change in 45% of lysosome related genes present in the KEGG database. Many of them were downregulated in Manf mzΔ96 embryos and some in Manf Δ96 larvae (Table 10; Additional file 9). The ATPase V-type H+ transporting subunit that maintains acidic environment in lysosomes showed downregulation in both mutants but was upregulated in Manf overexpressing larvae. The expression of other lysosomal membrane proteins and several lysosomal hydrolases was also altered.
Table 10

List of genes with altered expression according to microarray analysis related to KEGG lysosome pathway

KEGG

Gene

embryo

larvae

 

name

name

o.ex.

mut.

o.ex.

mut.

Description

AGA

CG10474

 

down

 

down

N4-(beta-N-acetylglucosaminyl)-L-asparaginase

SGSH

CG14291

  

up

down

N-sulfoglucosamine sulfohydrolase

AP-3

cm

  

up

down

carmine

ARS

CG7402

 

down

down

down

arylsulfatase B

ATPeV

CG7678

 

down

up

down

V-type H+-transporting ATPase subunit I

ATPeV

Vha100-1

up

down

  

Vha100-1

ATPeV

VhaSFD

 

down

  

Vacuolar H[+]-ATPase SFD subunit

cystinosin

CG17119

up

down

  

cystinosin

GUSB

CG2135

up

down

  

beta-glucuronidase

cathepsin

cathD

 

down

  

cathepsin D, pepsin A

LAMAN

CG6206

up

down

  

lysosomal alpha-mannosidase

CLN3

cln3

 

down

  

cln3

GLA

CG7997

 

down

  

alpha-galactosidase

ATPeV

Vha16

  

down

up

Vacuolar H[+] ATPase 16kD subunit

LYPLA3

CG31683

 

down

down

up

lysophospholipase III

CLN1

Ppt2

up

down

down

up

Palmitoyl-protein thioesterase 2

LAMAN

CG5322

up

down

down

up

lysosomal alpha-mannosidase

LYMP

trpml

 

down

down

up

control of membrane trafficking of proteins and lipids

NAGA

CG5731

up

down

up

up

alpha-N-acetylgalactosaminidase

LIMP

Tsp39D

  

down

up

Tetraspanin 39D

LIMP

Tsp29Fa

  

down

up

Tetraspanin 29Fa

HGSNAT

CG6903

  

down

up

heparan-alpha-glucosaminide N-acetyltransferase

saposin

Sap-r

  

down

up

Saposin-related

cathepsin

cathF

  

down

up

cathepsin F

LAMAN

CG9466

  

down

up

lysosomal alpha-mannosidase

LAMAN

CG9463

  

down

up

lysosomal alpha-mannosidase

AP-3

g

down

up

  

garnet, adaptor-related protein complex 3

clathrin

Chc

down

up

  

Clathrin heavy chain

GLB

CG9092

up

up

up

up

beta galactosidase

GTPase

Gie

   

up

N-terminally acetylated Arf-like GTPase

LAMAN

CG9465

   

up

lysosomal alpha-mannosidase

GBA

CG31148

up

up

 

up

glucosylceramidase

cathepsin

cathL

up

up

  

cathepsin L

GNS

CG18278

up

up

  

N-acetylglucosamine-6-sulfatase

GGA

Gga

up

up

  

Gga

AP-1

AP-1s

up

up

  

AP-1sigma

cathepsin

Cp1

up

up

  

Cysteine proteinase-1

LYMP

spin

up

up

  

spinster, lysosomal turnover regulator

GNS

CG30059

  

up

 

N-acetylglucosamine-6-sulfatase

Significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. ID stands for the particular homologue gene name in Drosophila. Notice that the same KEGG identifier can lead to several different genes. The according coloured scheme for lysosome KEGG pathway is presented in Additional File 9.

At the subcellular level, Manf colocalises partially with ER-targeted marker and very poorly if not at all with the lysosomal compartment (Figure 1F-H). Nonetheless, it is possible that the lack of Manf modifies the fusion of lysosomes with multivesicular body-like structures by some still unidentified mechanism.

Paternal rescue of the Manf mzΔ96 mutant embryos leads to reduction in the amount of differentially expressed genes

In Drosophila, substantial bulk of transcribed mRNAs and translated proteins necessary for early embryonic patterning and development are maternally contributed to the oocyte at high levels. These mRNAs and proteins are stored and used gradually during the embryogenesis and in some cases through the whole larval development. When the embryonic lack of maternal Manf was rescued by paternal wild type gene expression, the transcriptome changes were evident but restricted to a smaller number of genes than changes caused by the complete lack of Manf. As many as 98 genes were significantly upregulated by paternal rescue resulting in 18 functionally enriched GO term clusters (Additional file 10). We obtained GO terms related to response to stimulus and neurological process (8 genes), consisting of genes like transcription factor pros, pumilio (pum; encoding a mRNA binding protein involved in nervous system development), pastrel (pst; with unknown molecular function involved in memory and learning), Rop, rl, and small optic lobes (sol; calpain family peptidase). Transcripts of several genes coding membrane proteins also showed enrichment. Among them, we observed many genes coding ion-binding proteins (19%) such as klumpfuss (klu) and odd skipped (odd; DNA binding Zn-finger proteins important for embryonic nervous system development). Other enriched GO term clusters were membrane (7 genes) together with cell division, cell cycle and cytoplasm (9 genes).

Only 34 genes were downregulated by paternal rescue giving 4 functional GO term clusters (Additional file 11).

Ubiquitous overexpression of Manf results in transcriptional activation and upregulation of genes involved in cell cycle and cell death

We used enhancer trap line 69B-GAL4 to overexpress Manf, which we obtained as a commonly known GAL4 line with an epidermal expression pattern. After careful mapping the expression pattern of 69B-GAL4, we detected broad GAL4 expression in other tissues than epidermis - in central nervous system (non-glial), imaginal discs (wing and eye-antennal disc), garland cells, ring gland, but neither in fat body nor in gastric caeca. Ectopic expression of Manf under 69B-GAL4 rescues completely Manf Δ96 mutant lethality and the rescued adults are viable and fertile if maintained as a stock [5].

When comparing the gene expression profiles between Manf overexpressing and wild type larvae we found 614 genes upregulated that could be grouped in 102 functional GO term clusters (Additional file 12). This gene set showed enrichment in processes related to regulation of gene expression, protein localisation and transport, and cell cycle (e.g. kokopelli, an uncharacterized cyclin involved in stem cell maintenance and Retinoblastoma-family protein, the human Rb homolog). Genes involved in regulation of cell death were also upregulated (e.g. CG7188, a putative Bax inhibitor, rl, and klu). According to the previous study in HeLa cells, knockdown of MANF increased cell proliferation and susceptibility to ER stress induced cell death [7]. Our results support the involvement of Manf in regulation of cell cycle and cell death offering several candidate genes for further studies.

Manf overexpression in larvae caused downregulation of 340 genes annotated in 78 functional clusters (Additional file 13). The most prominent group consisted of GO terms such as membrane, plasma membrane, signal peptide, glycoprotein, disulfide bond, glycosylation site: N-linked (GlcNAc), integral to membrane, and transmembrane (77 genes). The majority of processes related to these GO terms take place in the ER such as cleavage of the signal peptide and disulfide bond formation. The main arthropod cuticular component chitin is composed of polymerised GlcNAc residues. Another prominent group was ion binding and metal binding (70 genes). Axon guidance, cell projection organization, neuron development, axonal defasciculation, cell motion, cell recognition (20 genes) were also enriched in line with our previous results implicating the role of Manf in neuritogenesis [5].

When comparing the upregulated genes in both paternally rescued embryos and in Manf overexpressing larvae, the common represented GO term clusters were ion binding (14 genes), membrane fraction (7 genes), oxidation reduction (8 genes) and cell cycle (5 genes) (Additional file 14). All together there were 57 annotated genes commonly upregulated by Manf, among these well known genes like Cbl, diaphanous (dia, formin, essential for actin-mediated events involving membrane invagination), Kinesin-like protein at 68D (Klp68D), rl, and Rop. Among the downregulated genes in both paternal rescue and Manf overexpression, there were only 6 genes in common e.g. CG34384, a diacylglycerol kinase involved in phosphoinositol signalling and glycerolipid metabolism. Conclusively, in Manf overexpression the typical growth factor signalling mediators rl and Cbl were upregulated. The links upstream of these mediators and downstream of secreted Manf still remain missing. The second cluster of genes was directly linked to membrane modifications and transport. Interestingly, the intracellular protein modification processes in ER were also enhanced by Manf overexpression. So presumably Manf has a dual role - one intracellularily in the ER, and the other extracellularly after being secreted.

Differentially regulated genes in larval Manf Δ96 mutant in comparison to Manf overexpression

Next, we searched for genes showing downregulation in Manf Δ96 larval mutant and were upregulated in Manf overexpression condition, and vice versa. Altogether 89 probes on the microarray showed this opposite regulation resulting in 62 annotated Drosophila genes (Additional file 15). In this group, there were genes responsible for locomotory behaviour (3 genes), genes involved in oxidation reduction and metal binding (10 genes), in cell division (4 genes), genes coding membrane proteins (7 genes), and genes involved in insulin signalling (2 genes). Because of the diversity of GO terms and low number of genes the functional annotation clustering did not give statistically significant results.

UPR and Parkinson's disease

Among the known genes involved in PD, 32 are conserved between mammals and Drosophila, and 44% of these were differentially expressed in our microarray assay (Figure 5, Table 11). Importantly, several genes from dopamine uptake (Dopamine transporter, DAT) and synthesis (ple coding for TH, Ddc, and punch) were differentially expressed (Table 2, Additional File 1). The expression of many genes involved in mitochondria and ubiquitin proteasome pathways were also altered. Increasing evidence indicates that organelle stress is a key event in neurodegeneration [14]. UPR is an adaptive process aiming to restore the cell homeostasis under ER stress conditions and to re-establish the properly folded protein synthesis. Under irreversible ER damage UPR initiates cell death pathway to eliminate damaged cells. Manf could be responsible for the recovery and survival pathway in UPR. In this scenario, the lack of Manf especially in the secretory cells with high rates of protein synthesis, including neurons with intensive neurotransmission, might result in shift from ER stress to UPR towards cell death. However, the mechanism how Manf dispatches this function is still unclear. Recently the structural homology between the Manf C-terminal folding and SAP domain of Ku70 has been demonstrated [34]. SAP domain of Ku70 is known to inhibit Bax-induced apoptosis [35] and in vitro experiments with mammalian cultured neurons showed that Manf rescues the NGF-deprivation induced cell death as efficiently as Ku70 itself [34]. On the other hand we showed that the overexpression of Manf resulted in upregulation of genes involved in oxidation reduction. The DA neurons are known to be very sensitive to oxidative stress because dopamine metabolites are highly oxidative compounds [36]. Therefore the upregulation of genes responsible for oxidation reduction could be already protective for DA neurons. Besides reactive dopamine metabolites, the mitochondrial dysfunction has been implicated in the neurodegeneration occurring in PD. We found that, in Manf mzΔ96 mutants with degenerating DA neurites, the nuclear genes coding for mitochondrial proteins were significantly enriched among the downregulated genes.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-134/MediaObjects/12864_2011_Article_4000_Fig5_HTML.jpg
Figure 5

Gene expression alterations in Manf mutants with a linkage to Parkinson's disease. A human disease KEGG pathway coloured with Drosophila homologues showing altered gene expression in Manf mutants - filled boxes with red represent genes that are upregulated, with blue downregulated expression or with purple differently altered gene expression between two Manf mutants. The unaltered components in the pathway are coloured green. Note that α-synuclein (SNCA) has no homologue in Drosophila. The complete list of altered genes is summarised in Table 11 with some additional genes not present in KEGG pathway but have been associated with PD elsewhere. The expression of genes with a role in dopamine uptake, intracellular transport, and synthesis is altered. Mitochondrial oxidative pathway complex I encoding genes show upregulation. The other mitochondrial proteins encoded by nuclear genes involved in PD such as Htra2 and DJ-1 expression is downregulated in Manf mutants. Some members of ubiquitin pathway are altered as well.

Table 11

List of genes with altered expression according to microarray analysis related to KEGG pathway of Parkinson's disease

KEGG

Human

embryo

larvae

Flybase

 

name

gene

o.ex.

mut.

o.ex.

mut.

ID

Description

TH

TH

 

up

  

CG10118

pale, tyrosine hydroxylase

VMAT

VMAT

   

up

CG6139

vesicular monoamine transporter

DAT

SCL6A3

up

down

  

CG8380

dopamine transporter

HTRA2

HTRA2

up

down

  

CG8464

HtrA serine peptidase 2

NDUFV1

NDUFV1

up

down

  

CG9140

NADH dehydrogenase

SNCAIP

SNCAIP

  

up

down

CG5424

forked, actin binding α synuclein interacting

CDCREL1

PNUTL1

  

up

down

CG7238

septin interacting protein 1

CytC

CYCS

  

up

down

CG14235

cytochrome-c oxidase

UQCRH

UQCRH

up

 

up

down

CG30354

ubiquinol-cytochrome-c reductase

 

SIP2

  

up

down

CG9188

septin interacting protein 2

 

SLC25A6

  

up

down

CG1683

mitochondrial adenine nucleotide translocase 2

Uchl1

UCHL

  

up

 

CG4265

Ubiquitin carboxy-terminal hydrolase

mPTP

VDAC2

up

up

up

down

CG17137

Porin2, voltage-gated anion channel

ATPase

ATP5EP2

  

up

 

CG31477

mitochondrial ATPase ε subunit, F1 complex

NDUFA2

NDUFA2

  

up

up

CG15434

NADH dehydrogenase

 

SEP4

 

up

 

up

CG9699

Septin 4

Ube2J2

UBE2J2

  

down

up

CG5823

ubiquitin-protein ligase

NDUFV2

NDUFV2

   

up

CG5707

NADH dehydrogenase

 

UBE2H

   

up

CG2257

Ubc-E2H, ubiquitin-protein ligase

Ube2L3

UBE2L3

  

down

down

CG12799

Ubiquitin conjugating enzyme 84D

 

UBQLN1

  

down

 

CG14224

EF1B ubiquitin-assoc. translation elongation factor

Ubh4

UCHL

   

down

CG3431

Ubiquitin C-terminal hydrolase

 

USP36

   

down

CG5505

scrawny, ubiquitin protease

 

SIP3

   

down

CG1937

septin interacting protein 3

Uba1

UBA1

 

up

  

CG1782

ubiquitin activating enzyme 1

CytC

CYCS

up

up

  

CG2249

cytochrome-c oxidase

 

SEP1

 

up

  

CG1403

Septin-1

 

UBE2E2

 

up

  

CG6720

Ubiquitin conjugating enzyme 2

DJ-1

DJ-1

 

down

  

CG6646

DJ-1α

CytC

CYCS

 

down

  

CG13263

cytochrome c distal

CytC

CYCS

 

down

  

CG14028

cyclope, cytochrome-c oxidase

CytC

CYC1

 

down

  

CG4769

electron transporter

ATPase

ATP5G2

down

down

  

CG1746

hydrogen-exporting ATPase

ATPase

ATP5E

 

down

  

CG9032

stunted, hydrogen-exporting ATPase

Significant alterations in gene expression from wild type are shown by word code; "up" represents upregulation and "down" downregulation of gene expression. Notice that the same KEGG identifier can lead to several different genes. Several Drosophila genes associated with processes in PD missing from KEGG pathway were added. The according coloured scheme for Parkinson's disease KEGG pathway is presented in Figure 5.

Conclusions

We have studied the gene expression changes of two slightly different Manf mutants in Drosophila. Surprisingly, the expression profiles of embryonic lethal Manf mzΔ96 and larval lethal Manf Δ96 mutants were quite diverse. It might be due to the dissimilar roles of Manf gene during embryonic and larval stages or it indicates the difference between the absolute lack of Manf versus gradual diminishing of maternally contributed stores of Manf gene products. Our microarray analysis followed by functional annotation clustering revealed statistically significant enrichment related to metabolism and membrane transport and transporters. The observed changes in membrane traffic were supported by ultrastructural studies of Manf mzΔ96 mutant. More than 40% of known Drosophila genes related to ER and UPR showed altered expression levels in Manf mutants. We found changed expression of several genes known to be associated with processes altered in PD such as oxidative phosphorylation, mitochondrial function, dopamine metabolism and uptake, and protein ubiquitinaton. The lack of Manf in Manf mzΔ96 mutant embryos resulted in massive upregulation of stress, defense, and immune response related genes as well as genes involved in proteolysis and cell death. Overexpression of Manf resulted in upregulation of genes involved in oxidation reduction, an important process to protect DA neurons from oxidative stress. Thus, our results support the previously reported findings in mammalian cells that upregulation of Manf is important in UPR and could be protective for the cell. It is also evident that ER stress leads to UPR and cell death in the absence of Manf. These effects were less drastic when Manf was gradually abolished in Manf Δ96 mutant larvae and related to several metabolic processes and downregulation of genes involved in replication, transcription and splicing. Still, stress and defense related genes were enriched among the upregulated genes of both Manf mutants.

Methods

Fly strains

Drosophila melanogaster adults were maintained at 25°C on malt and wholemeal flour based standard food, for egg laying apple juice agar plates with yeast paste were used. Wild type flies were w - , all genotypes used were of w - background: hs70Flp/+;; FRT 82B Manf Δ96 /Manf Δ96 , Manf Δ96 /TM3 Sb Ser twi > GFP. For ectopic overexpression 69B- GAL4 [37] and UAS-DmManf 133 (3rd chromosome insertion) were used. As an ER marker ER-targeted sqh-EYFP flies were used. UAS-GFP-rab5, UAS-GFP-rab11, UAS-chc-GFP, and UAS-GFP-rab7 were obtained from C. Samakovlis and ppl > GAL4 from V. Hietakangas. Other fly lines were obtained from Bloomington Drosophila Stock Center or generated by us [5].

RNA isolation

For RNA extraction, embryos were collected from apple juice plates, washed with embryowash, dechorionated by standard bleach treatment and washed thoroughly. Both embryos and larvae were separated and picked by phenotype and staged under microscope onto fresh apple juice plates, collected into Eppendorf tubes and snap frozen. Total RNA was extracted with Qiagene RNAeasy extraction Kit (Qiagene) according to manufacturer's recommendations, treated with RNase-free DNase (Promega) 15 min at 37°C and purified by RNA clean-up kit (Macherey-Nagel). RNA was quantified using a NanoDrop-1000 spectrophotometer and RNA quality was monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies).

Microarray experiments

For microarray experiment three biologically independent samples for each genotype were used. Diluted spike controls (Agilent) were added to 1 μg of total RNA samples and in vitro transcribed and labeled with Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion/Applied Biosystems). The dyes used were cyanine 3 (Cy3), cyanine 5 (Cy5) or Alexa 488, as previously described [38]. Dye incorporation and received aaRNA yield were monitored by the NanoDrop ND-1000 Spectrophotometer.

Hybridisation

5 μg of each differentially labelled aaRNA was fragmented at 60°C for 30 min in a reaction volume of 55 μl containing Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent's fruit fly Microarray Kit 4x44k, P/N G2519F (Agilent Microarray Design ID 018972) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation. The slides were then scanned by Axon 4200AL scanner.

DNA microarray analysis

The microarrays images were segmented and the median intensity of each spot was estimated by the software GenePixPro® 6.0 (Axon). The data were then imported into R software http://cran.r-project.org/ and preprocessed by the BioConductor package Limma [39]. Linear model followed by moderated t-test was utilized for finding the differentially expressed genes (P-value < 0.01 after Benjamini and Hochberg correction) between Manf Δ96 , Manf mzΔ96 , Manf mΔ96 /+, 69B > Manf 133 and w - . Lists of significant genes were screened by the DAVID 6.7 annotation tools [40, 41] in order to find over-represented biological themes. Default DAVID parameters were used. To identify the pathways altered, the online tool available from Kanehisa laboratories, KEGG Mapper was used [42]. All microarray data are MIAME compliant and available at the NCBI GEO database (ID GSE28820).

Quantitative PCR

For qPCR, independent biological samples were used for RNA extraction. 4 μg of total RNA was reverse transcribed with MMLV reverse transcriptase (Promega) according to manufacturer's instructions using oligo(dT) primers. The primers were designed with the help of Universal ProbeLibrary Assay Design System (Roche Applied Science) and are listed in Additional file 16. qPCR was performed with Lightcycler 480 real-time PCR system (Roche Diagnostics) with the help of pipeting robot Robotics4 (Corbett Robotics) on 384-well plates using Lightcycler 480 SYBR Green I Master complemented with 5 pmol of primers and cDNA corresponding to 40 ng of total RNA used in reverse transcription. Three replicates for each reaction were included in the PCR runs. Results were analysed with Lightcycler software version 1.5.0.39.

Transmission electron microscopy and immunohistochemistry

The embryos for TEM were treated as previously described [43]. The whole larvae were subjected to high pressure freezing to visualise the cuticle layers as described earlier [44]. The primary antibodies used were rabbit phospho-eIF2α (Ser51) antibody (1:1000, Cell Signaling Technology), mouse α-DmTubulin (1:1000, Sigma) and rabbit α-DmManf (1:1000) [5]. Immunohistochemistry and imaging were performed as previously described [5]. To visualise the lysosomes, Lysotracker Red DND99 (Invitrogen) was used. The red colour of Alexa568 dye was changed to magenta in order to help colour blind people to distinguish it in the combinations with green.

Western blotting

For Western blotting about 100 embryos of stage 17 were collected, genotyped, and homogenised in 10 mM HEPES, 1 mM EDTA, 0.25 M sucrose homogenising buffer, pH = 7.3 in the presence of protease inhibitor cocktail (Complete Mini, Roche). The concentration of proteins was measured with Bio Rad protein assay DC reagents. The equal amounts of total protein were mixed with 3 × Laemmli loading buffer and boiled at 99°C for five minutes. Up to 6 μg of total protein were loaded per lane to SDS-acrylamide gel. Western blotting was further proceeded according to the standard manufacturer's instructions (Amersham Biosciences). For the quantification of Western blotting results ImageJ analysis software was used. Quantification was based on area measurements and intensity calculations in comparison with the anti-tubulin loading control.

Declarations

Acknowledgements

We thank Mart Saarma, Päivi Lindholm, Osamu Shimmi, Christos Samakovlis and Ville Hietakangas for reagents and fly stocks, Eeva-Marja Turkki for the technical assistance, Jouni Kvist, Panu Somervuo, Petri Törönen, and Ilya Plyusnin for the comments and help with microarray analysis, Mikko Airavaara for the help with Western blot quantifications, Juha Partanen and Johan Peränen for critical comments during the writing of the manuscript. This work was supported by the Academy of Finland Neuroscience Program, the Michael J. Fox Foundation, the Finnish Parkinson Foundation and the University of Helsinki research funds.

Authors’ Affiliations

(1)
Department of Biosciences, University of Helsinki
(2)
Institute of Biotechnology, University of Helsinki
(3)
Department of Bioscience and Nutrition, Karolinska Institutet

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