Department of Biological Sciences (BIO)

Master theses submitted in 2015

Alba Kaci

Alba Kaci (I. Aukrust, L. B. Gundersen, P. R. Njølstad)

SUMOylation of hepatocyte nuclear factor-1 alpha (HNF-1A). Its possible role for transcription factor function

Hepatocyte nuclear factor-1 alpha (HNF-1A) is a transcription factor which regulates the expression of several liver-and pancreas-specific genes. Mutations in the HNF1A gene, encoding HNF-1A, are the most common cause of Maturity-onset Diabetes of the Young (MODY3; HNF1A-MODY), a monogenic form of diabetes associated with autosomal dominant inheritance, young -onset (before 25 years of age) and pancreatic β-cell dysfunction. The cellular mechanisms for HNF-1A regulation of target genes are poorly investigated and in particularly how post translational modifications control the protein function remains largely unknown. SUMOylation is a post-translational modification (PTM) engaged in many cellular processes, where the regulation of transcription factors is the best characterized.

This study shows that HNF-1A is SUMOylated in cellulo at Lysine 46 and Lysine 506, and that its level of SUMOylation is increased by the SUMO E3 ligase; protein inhibitor of activated STAT (PIASγ). Luciferase reporter assays in MIN6 β-cells demonstrated that HNF-1A transactivation of the rat albumin promoter was repressed when co-transfecting with human SUMO-1 and UBC9 (E2). In addition, co-transfecting with the E3 ligase PIASγ repressed the transactivation of HNF-1A even more. In addition to a repressing effect, PIASγ was shown to stabilize HNF-1A in cellulo, most likely in a conformational state inadequate for full HNF-1A transactivating function. In contrast, SUMOylation of HNF-1A seems not to affect the sub-cellular localization and DNA binding ability of the transcription factor. Taken together, these results indicate that SUMOylation is largely involved in the regulation of HNF-1A function. This may have possible implications for HNF-1A regulation of glucosestimulated insulin secretion.

Henriette Wangen

Henriette Wangen (R. Male, L. Sandlund)

Charachterization of Fushi Tarazu Factor 1 in the Salmon Louse (Lepeophtheirus salmonis)

The salmon louse, Lepeophtheirus salmonis, is a crustacean ectoparasite harming both wild and farmed salmonid fish such as Atlantic salmon (Salmo salar). The aquaculture industry is facing major challenges to control sea lice infestations, generating financial losses and causing impacts to the welfare of the fish. Current treatments against sea lice are shown be ineffective over time in many places as the lice develop resistance and tolerance. This has led to the need for new treatment methods to fight the sea lice based on knowledge at its molecular level.

Nuclear receptors (NRs) are transcription factors involved in many important biological processes. NRs may hence be suitable targets for the development of new treatment methods that can be used to inhibit vital processes of development in the sea lice. In arthropods, NRs mediate many vital processes via the ecdysteroid signalling pathway. In Drosophila, the orphan NR, Fushi Tarazu Factor 1, is induced immediately after decline in 20E levels, and has been found to have important functions during development, reproduction and molting. Furthermore, in the ecdysone-signalling pathway, expression of FTZ-F1 has been found necessary for activation and inhibition of down-stream targets.

The life cycle of L. salmonis consists of eight developmental stages, each separated by a molting event. In situ hybridization revealed FTZ-F1 transcript in all tissues of copopodid larval stage, in the ovaries and subcuticula of adult females and in adult male spermatophores. FTZ-F1 knockdown by RNAi in L. salmonis showed substantial differences in phenotypes based on whether the transcript was positioned in the A/B domain, just before the DNA-binding domain (FTZ-F1 fragment 1), or from the ligand-binding domain and into the untranslated region (FTZ-F1 fragment 2). FTZ-F1 (fragment 1) knockdown in larvae resulted in leg-and tail abnormalities in one RNAi experiment and pebbled and uneven exoskeleton in another. Knockdown in pre-adult II did not result in any clear abnormalities. In contrast, FTZ-F1 (fragment 2) knockdown in larvae caused molting-arrest and what appeared to be nauplius II larvae with similar morphology as a copepodid. Knockdown in pre-adult II also obstructed the development of egg-strings. These results suggest that FTZ-F1 in L. salmonis has a broad expression pattern in tissue and development during larval stages and may be involved in reproduction during later stages of development.

Hulda María Harðardóttir

Hulda María Harðardóttir (M. Ziegler, R. H. Skoge)

Identification of the DNA sequence encoding Neurospora crassa NADase and characterization of the recombinant protein

Neurospora Crassa (N.crassa) is a bread mold that has been used as a model organism inmolecular biology since the late 1920s. N.crassa NADase is an enzyme that catalyze theconversion of NAD+ to nicotinamide and ADP-ribose. The enzyme was first partially purified in1951 from mycelial N.crassa grown in zinc-deficient medium. The enzymatic properties of theenzyme was in the following years subjected to much study. The enzyme is intriguing, as it haspure hydrolysis activity, which is rare among eukaryotic NADases. N.crassa NADase is known tobe regulated during development, however, its biological function is still unknown. Also, eventhough the enzyme has been known for decades, the only known information about the genomicsequence is that it is found on linkage group IV.

In this study, the DNA sequence encoding N.crassa NADase was identified throughalignments of peptide sequences covering parts of the full-length NADase to translated N. crassagenome sequences. The identified DNA sequence is found on linkage group IV in the N.crassagenome and is described as NCU07948 hypothetical protein in NCBI. This gene has no sequenceidentity with known NADases such as human CD38 or Aplysia californica cyclase. The NCU07948sequence was indeed found among mRNA transcripts in N.crassa. The cDNA sequence ofNCU07048 was codon optimized for human expression and cloned into an expression vector. Thesame sequence was also cloned into prokaryotic expression vectors for expression of recombinantncNADase in the cytoplasm and periplasm of E. coli cells. All resulting recombinant proteins werefunctional NADases with pure hydrolysis activity. The recombinant N.crassa NADases (ncNADase)hydrolyzed both NAD+ and NADP+, did not exhibit any ADP-ribosyl cyclase activity or base exchangeactivity, and were insensitive to inhibition by nicotinamide. All these observations matchwith the already known properties of endogenous N.crassa NADase.

ncNADase expressed in human cells was expressed as a secreted N-glycosylated protein.Purified human ncNADase-FLAG had a KM value of 27 μM for ε-NAD+ and a VMAX value of 0.35μmol/min*mg. The enzymatic activity increased by ~ 50% with the addition of 2.5 mM ZnCl2, whileit was reduced to ~ 34 % by the addition of 2.5 mM EDTA. The major difference between therecombinant ncNADases expressed in human cells and E. coli cells, was that the non-glycosylatedE. coli ncNADases were more sensitive towards higher temperatures. In conclusion, the presentstudy finally revealed the genome sequence encoding for N.crassa

Ina Blindheim Johansen

Ina Blindheim Johansen (A. Goksøyr, M. Eide, R. Lille-Langøy, O.A. Karlsen)

Ligand aktivering av rekombinante vitamin D reseptorer, samt kloning og uttrykking av cyp24a1 fra atlanderhavstorsk (Gadus morhua)

Det har blitt vist at en rekke stoffer som frigjøres til miljøet som et resultat av menneskelig aktivitet har negative effekter på levende organismer. Mange av disse fremmedstoffer brytes ned i organismene gjennom prosesser som samlet kalles biotransformasjon. Biotransformasjonen reguleres blant annet av kjernereseptorer, en gruppe transkripsjonsfaktorer som også er målmolekyler for en rekke miljøgifter.

Denne masteroppgaven fokuserer på en av reseptorene i NR1IVgruppen, vitamin D reseptor (Vdr, Nr1i1). De to øvrige medlemmene i NR1I-gruppen, konstitutiv androstan reseptor (CAR, NR1I3) og pregnan X reseptor (PXR, NR1I2) er betraktet som xenosensorer da de kan aktiveres av et bredt spekter av endogene og eksogene stoffer, som initiere transkripsjonen av gener involvert i alle faser av biotransformasjonen.  

I 2011 ble genomsekvensen til atlanterhavstorsken (Gadus morhua) sekvensert. I etterfølgende bioinformatiske analyser av genomsekvensen og kloningsforsøk er pxr ikke identifisert. Genomsekvensen inneholder derimot to gener som koder for vdr i atlanterhavstorsk.  Med utgangspunkt i det tilsynelatende fraværet av pxr og tilstedeværelse av to vdr paraloger i torsk sammen faktumet at CAR er fraværende i fisk, er det derfor interessant å undersøke hvordan Vdr blir påvirket av f.eks miljøgifter.

Agonistforsøket med Vdr-paralogene i atlanterhavstosk har tidligere blitt utført i gruppen (Ueland E., 2013) med 12 utvalgte endo- og eksogene stoffene som er vist å aktivere andre kjernereseptorer i NR1I-gruppen.  Resultatet fra agoniststudien viste at kalsitriol, som forventet, var en kraftig Vdr-agonist.  Enkelte stoffer, som blant annet heksabromosyklododekan (HBCD) og diklordifenyltrietan (DDT), førte til nedgang i aktiveringen av Vdr reseptorene. Dette dannet grunnlaget for antagonist studiet utført i denne oppgaven, hvor hovedarbeidet er å undersøke hvordan aktiveringen av Vdr blir påvirket av kalsitriol i kombinasjon med 11 andre ligander brukt i agoniststudiet. Samtidig var det av interesse å undersøke Vdr-paralogenes følsomhet for kalsitriol sammenliknet med tilsvarende ortologer fra sebrafisk. Dessuten var det et mål å klone og rekombinant uttrykke atlanterhavstorsken cyp24a1.

Ut ifra arbeidet gjort i oppgaven kan det konkluderes med at Vdrtorsk har smal ligandspesifisitet, da kun kalsitriol var vist å virke som agonist med en tydelig dose-respons sammenheng.  Antagoniststudiene indikerte at 9-cis retinsyre kunne kanskje IV virke som en antagonist for VdrVparalogene i atlanterhavstorsk, mens blant annet HBCD og DDT antydet synergistiske effekter ved lave konsentrasjoner av disse ligandene. 

Vdr reseptorene i atlanterhavstorsk er vist å være mindre følsomme for kalsitriol enn tilsvarende kjernereseptorer i sebrafisk.  Dette observasjonen kan muligens forklares ut i fra det høye kalsitriol innholdet som tidligere er observert i torsk, samt observerte forskjeller i ligandbindingsdomenet i Vdr reseptorene mellom torsk og sebrafisk.

Ett av Vdr-metabolismen målgen, cyp24a1, fra atlanterhavstorsk ble klonet, sekvensert og rekombinant  uttrykkt  i  E. coli som  et funsjonproteinet  bestående  av  maltosebindende  protein  og  6xHis-tag. Ut fra dette kan vi konkludere at Cyp24a1 er tilstede i atlanterhavstorsk.

Ingvild Stensland

Ingvild Stensland (R. Aasland, S. Värv)

Brd4 as a candidate protein for the recognition of histone H3 lysine 27 acetylation

Post-translational modifications (PTMs) of histone tails are associated with the regulation of transcriptional processes. Patterns of histone modifications mark regions of high or low transcriptional activity, and are recognized by “reader proteins”. The reader proteins are often transcription factors that recruit chromatin remodelling complexes or enzymes that add further modifications to histone tails.

Genomic enhancer regions, responsible for cell-type specific activation of different genes, have been found to exhibit distinct patterns of histone modifications. Active enhancers are enriched in acetylation of histone H3 lysine 27 (H3K27ac), a mark which distinguishes active from poised and primed enhancers. In this work, we wished to find a protein which specifically recognizes this histone modification.

Bromodomain- containing protein 4 (Brd4) is a transcriptional regulator that is known to be involved in the regulation of elongation by RNA polymerase II (Pol II). Brd4 has been shown to co-localize with H3K27ac at active enhancers, and was therefore chosen as a candidate for selective recognition of H3K27ac.

Possible binding between the two bromodomains of Brd4 and H3K27ac was investigated by histone peptide binding assays, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Brd4 bromodomain constructs containing one or both bromodomains were engineered, and proteins were expressed and used in binding assays. Strong binding between Brd4 bromodomain 1 (BD1) and a H4 peptide acetylated at four lysines (H4tetra-ac) was observed, but in contrast to a previously published study, no binding between either of the Brd4 bromodomains to H3K27ac was detected in histone peptide binding assays and ITC.

Possible reasons for these discrepancies are discussed and alternative functions for Brd4 and its bromodomains at enhancer and promoter regions are suggested.

Karina Dale

Karina Dale (S. Ellingsen, J. Rasinger)

Vitamin E Modulates Toxic Effects of Endosulfan in Zebrafish (Danio rerio) Embryos

Aquaculture is an important and among the fastest growing food-producing industries worldwide. The rapid growth of aquaculture has generated a need for the discovery of new ingredients for fish feed, to avoid overexploitation of marine resources that earlier dominated the feed composition. Vegetable-based ingredients are one of the main replacements for marine products in aquaculture feeds. While marine ingredients may be contaminated with elevated levels of persistent organic pollutants (POPs), vegetable ingredients may introduce other contaminant types to the feeds including traces of agricultural pesticides. In recent years, it has been confirmed that induction of oxidative stress is part of the toxic mechanism of many pesticides. Cells have several defense mechanisms for protection against oxidative stress, involving use of both endogenous enzymes and nutrients from food. However, if the impact on this system is great enough, the defense mechanisms will be overwhelmed, resulting in cellular damage. The interactions of antioxidant nutrients and toxic compounds can modulate the degree of the final toxicity.

To gain more knowledge on interaction effects of pesticides and antioxidants on pesticide-induced toxicity in early life stages, a modification of the zebrafish embryotoxicity test (ZET) was utilized as a model system. Zebrafish (Danio rerio) embryos were exposed at 6-72 hpf to either endosulfan (ESF) or vitamin E (α-TOC) alone, or a combination of these two (co-exposure). Phenotypic studies at 48 hpf showed that ESF led to a dose-dependent increase in embryonic deformities, in terms of axis malformations, pericardial edema and reduced pigmentation. The co-exposure with both ESF and α-TOC showed that higher concentrations of α-TOC (1-3 mM) significantly (p<0.05) reduced ESF-induced embryonic malformations. Embryos exposed only to α-TOC exhibited stable survival rates and a low degree of malformations in all groups, suggesting that the utilized α-TOC concentrations had a negligible effect on the embryos. Moreover, behavior studies showed that ESF caused hyperactivity in 5 dpf zebrafish larvae, indicating a neurotoxic effect of ESF. The behavioral changes were ameliorated by 3 mM α-TOC. Additionally, high ESF concentrations caused down-regulation of the antioxidant genes cuzn-sodgpx1a and cat, in addition to cyp1a1, indicating that ESF promoted oxidative stress in the embryos. α-TOC was not able to prevent this down-regulation. At lower ESF concentrations, the expression levels of the genes cuzn-sodgpx1acat and cyp1a1 were dose-dependent. Together, these results indicate that α-TOC to some extent protects against ESF-induced phenotypes and behavioral changes. However, the effect of the ESF concentrations utilized still superseded the antioxidant gene expression in presence of α-TOC.

Sukarna Kar

Sukarna Kar (H. C. Seo, R. Male)

Study of Ecdysone receptor and Ultraspiracle in the salmon louse (Lepeophtheirus salmonis)

The salmon louse (Lepeophtheirus salmonis) is a parasite living on mucus, skin and blood of salmonids fishes. L. salmonis causes lesions and infections on fish fins and skins and such physical damages often lead to other diseases. The global salmon farming industry faces huge economic losses caused by the prevalence of salmon lice and is struggling to contain frequent salmon lice outbreaks. Chemical treatments have been a traditional way to combat salmon lice problem, but increased resistance of salmon lice to currently available chemicals leave the salmon aquaculture communities with fewer options. Therefore, it is warranted to search for new, efficient and environment-friendly drugs which are based on molecular studies of nuclear receptors of salmon lice. Investigation of ecdysone receptor (EcR), which acts as a receptor for the ecdosteroid hormone, is one of such molecule-based new drug searches. The ecdosteroid hormone plays an important role during molting, maturation and reproduction processes of crustaceans. Ecdysteroid agonists for EcR that disrupt these processes could be novel pesticides to control salmon lice.

In this study, expression constructs of L. salmonis ecdysone receptor (EcR) and ultraspiracle (USP), which forms a heterodimer with EcR, were made and they were expressed in E. coli. The EcR constructs (both ligand-binding domain and full-length) were expressed well, but the full-length USP construct was not expressed. Immobilised metal ion affinity chromatography (IMAC) was used to purify EcR proteins. The two EcR proteins, i.e., ligand-binding domain (LBD) and full-length EcR, bound very poorly to the Ni-resin. The reason can be that the 6x His tag was buried inside of the MBP-attached EcR protein, thus it was not available to the Ni-resin. To circumvent this challenge, ion-exchange chromatography (IEC) was employed. At a very low salt concentration (6.7 mM NaCl), the EcR proteins were eluted as flowthrough, whereas much of impurities remained in the column, hence achieving substantial purification. As the last step of purification, size exclusion chromatography (SEC) was used. The proteins were eluted at near the void volume, suggesting they are in a form of aggregates under the experimental conditions. With partially purified EcR-LBD, a binding study between EcR-LBD using isothermal titration calorimetry (ITC) was attempted.

Kristina Strand

Kristina Strand (T. H. Røst, A. J. Svärd, J. Fernø)

ORMDL3 plays a potential role in insulin signalling in adipocytes

A previously performed microarray, analysing global gene expression in subcutaneous adipose tissue of patients before and one year after bariatric surgery, identified ORMDL3 as being upregulated after surgery and to correlate negatively with BMI and markers of insulin sensitivity, such as HOMA-IR and TG/HDL ratio. Additionally, ORMDL3 was found to be enriched in the adipocyte fraction of the adipose tissue, suggesting that elevated levels of ORMDL3 may have a beneficial role in metabolic homeostasis. ORMDL3 is known to play a role in ceramide homeostasis by negatively regulate serine palmitoyltransferase, the ratelimiting enzyme of the de novo ceramide biosynthesis. Ceramide is a sphingolipid found to impair insulin signalling by deactivating or inhibiting the activating phosphorylation of Akt/PBK, a protein kinase important in the insulin-signalling pathway. Levels of ceramide are found to be elevated in the adipose tissue of obese individuals and degradation of ceramide in adipose tissue is found to prevent systemic insulin resistance. The role of ORMDL3 as a regulator of de novo ceramide biosynthesis has been described in other cell types with asthma as the clinical focus, but its role in adipocytes has not yet been studied.

In this study, the effect of modulating levels of the ORMDLs on insulin signalling and glucose uptake in human primary adipocytes and 3T3-L1 adipocytes was investigated. The gene expression level of ORMDL3 during differentiation was analysed in two different adipocyte cell lines as well as in human primary adipocytes. Additionally, adipocytes were treated with palmitate and LPS to analyse the effect on insulin signalling measured by levels of pAkt. Finally, the relationship between ORMDL3, ceramide and insulin signalling was investigated in human primary adipocytes by the use of the ceramide analogue, C2-ceramide.

ORMDL3 seemed to be upregulated during adipocyte differentiation. Knockdown of ORMDL3 was successful in reducing gene levels of ORMDL3, however only minor effects of knockdown could be observed on pAkt-levels. Palmitate, the substrate of ceramide biosynthesis, did not seem to affect glucose uptake in 3T3-L1 adipocytes. Our results indicate that palmitate increased pAkt, while pAkt was decreased when palmitate was co-treated with the inflammatory signal LPS. ORMDL3-mediated regulation of ceramide biosynthesis seems to be of relevance in adipocytes, with ORMDL3 siRNA sensitizing the cells to a small dose of C2-ceramide.

This study confirmed that ORMDL3 in WAT is upregulated after bariatric surgery and that it is enriched in adipocytes. Despite the difficulties in establishing a specific function of ceramide, ORMDL3 and pAkt after treatment with palmitate, LPS and myriocin, we were able to establish a relationship between ceramide, the ORMDLs and pAkt in human primary adipocytes. This indicates that ORMDL3 plays a likely role in feedback inhibition of de novo ceramide biosynthesis also in adipocytes, which might be relevant for the endocrine function of adipose tissue in obesity and associated metabolic diseases.

Henriette Didriksen

Henriette Didriksen (T. Arnesen, L. M. Myklebust, H. Foyn)

Inhibition of human N-terminal Acetyltransferases by bisubstrate analogues

N-terminal acetyltransferases (NATs) are enzymes catalysing the acetylation of the Nterminal α-amino group of proteins. N-terminal (Nt) acetylation is a protein modification observed in 80-90% of human proteins, a process mostly occurring co-translationally. Currently, a group of six NATs (NatA-F) are known in human. Each NAT processes a distinct population of mostly proteins based on the specific sequence of their N-termini.

NATs may act as oncoproteins in human cancer cells and depletion of these enzymes induce cell-cycle arrest, apoptosis or autophagy in cancer cells. In light of this knowledge, several NAT-inhibitors were developed which may act as a starting point for direct or indirect anticancer therapeutics. The inhibitors contain a peptide part mimicking the substrate specificity of the NATs and are designed to compete with substrate (Ac-CoA) binding of the active site.

A range of inhibitors directed to the different NAT-complexes were tested for their potency and effect. First, all inhibitors were tested on the NAT-complexes they were developed towards. Next, these inhibitors were tested towards other NAT-complexes to analyse their specificity. The most efficient inhibitors developed and tested for the different NATs in this study were as follows: CoA-Ac-SSME (IC50 = 1.28 µM ± 0.18 µM) for NatA; CoA-Ac-MDEL (IC50 = 1.13 µM ± 0.14 µM) for NatB; CoA-Ac-EESL (IC50 = 32.12 µM ± 3.71 µM) for hNaa10; CoAAc-MLEP (IC50 = 0.62 µM ± 0.06 µM) for hNaa50; and CoA-Ac-MAPLDLD (IC50 = 0.73 µM ± 0.06 µM) for hNaa60.

As mentioned, these inhibitors may be potential therapeutic agents in cancer therapy. However, since these synthesised NAT-bisubstrate analogues are not themselves cellpermeable inhibitors, different nanodevices able to transport small molecules through the membrane were tested. By fluorescence spectroscopy, nanoparticles (NP) were found more efficient than liposomes to penetrate the membrane of HeLa cells. However, more elaborate testing of the NP capacity to deliver NAT-inhibitors is necessary.

Ida Marie K. Lavik

Ida Marie Kjærefjord Lavik (B. B. Johansson, K. Fjeld, P. R. Njølstad, A. Molven)

The effect of CEL in pancreatic disease: A study on secretion, endocytosis and cell viability

Carboxyl ester lipase (CEL) is a digestive enzyme mainly expressed in the pancreatic acinar cells. Two pathogenic variants of CEL have been discovered. A frame shift mutation in the CEL gene causes a dominant inherited disease characterized by pancreatic exocrine dysfunction and diabetes. The mutant protein (CELMUT) has a higher tendency to aggregate as compared to the wild-type protein (CEL-WT), and increased endocytosis of CEL-MUT is detected in both acinar- and β-cells of the pancreas. In addition, a hybrid deletion allele (CEL-HYB) of the CEL locus has previously been identified as a novel genetic risk factor for chronic pancreatitis. Expression of CEL-HYB in cellular models suggests a disease mechanism involving impaired secretion and intracellular accumulation of the protein.

In this study, we aimed to further investigate the disease mechanisms of CEL-MUT and CEL-HYB. Given that all patients identified so far are heterozygous carriers of CEL-MUT or CEL-HYB, we wanted to analyse the effect of these variants on the normal CEL protein. In HEK293 cells, we found that CEL-MUT increased the secretion rate of CEL-WT whereas CEL-HYB had the opposite effect: CEL-WT was retained inside the cell. Furthermore, we observed for the first time that CEL-MUT was aggregating in the cell pellet together with the CEL-WT protein. We also identified aggregating CEL-HYB protein in the same fraction.

Since digestive enzymes are transported via pancreatic ducts on their way to the stomach, we wanted to analyze the uptake of CEL in pancreatic ductal cells (PANC-1). Endocytosis in PANC-1 cells showed the same trend as previously detected in other cell models: CEL-HYB was taken up more strongly than the CELWT, but less than CEL-MUT. However, prolonged incubation time was required for endocytosis in PANC-1 cells as the maximum level of uptake was detected after 24 h incubation, not 30 minutes as previously reported for other cell types. Internalization of CEL-MUT or CEL-HYB resulted in both reduced viability and increased apoptosis of the PANC-1 cells.

Taken together, this study provides new information about the underlying pathological mechanisms of CELMUT and CEL-HYB. An important finding is that the pathogenic variants of CEL could have an effect on the CEL-WT protein. Thus, it is recommended to study the pathogenic CEL variants together with the WT protein.

Helene T. Bevan

Helene Torkildsen Bevan (K. E. Fladmark, A. K. Frøyset)

Neuronal DJ-1 functions in a zebrafish model for Parkinson’s Disease

Parkinson’s Disease is a neurodegenerative disease characterised by a progressively degrade and eventually death of dopaminergic neurons in the substantia nigra. Our group focuses on DJ-1, a protein associated with an autosomal recessive early onset form of Parkinson’s Disease, in which the gene encoding DJ-1 has either a knockdown or loss-of-function mutation. DJ-1 seems to function as a redox sensitive sensor and a general protector of oxidative stress.

This thesis aims to study and characterise a zebrafish line with neuronal overexpression of DJ-1. This was done in terms of both its phenotype and its responses to oxidative stress. Linking to our overall aim, we want to broaden our understanding of DJ-1’s role in neurodegenerative diseases and neuroprotection. As a long-term goal, we want to determine the cell-specific differences between glial and neuronal DJ-1 overexpression.

We successfully established a transgenic zebrafish with neuronal DJ-1 overexpression. This transgene was found to be phenotypically similar to the wild type, although evidence shows that neuronal DJ-1 effects the overall protein expression. However, none of the proteins were identified. Transgenic larvae exposed to rotenone were largely protected from rotenone-induced death and injury. We also found changes in the protein profiles that indicates different responses to oxidative stress between wild type and our transgene.

The results presented in this thesis indicate that neuronal DJ-1 is important in neuroprotective mechanisms.

Sandra K. Østerbø

Sandra Kleppe Østerbø (K. E. Fladmark, A. K. Frøyset)

Effects of β-methylamino-L-alanine (BMAA) on brain-derived cell lines and zebrafish

β- methylamino-L-alanine (BMAA) is a neurotoxin produced by cyanobacteria. This compound has been described as a possible cause of the spontaneous forms of amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD) and also the neurodegenerative disease amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). The mechanism behind BMAA toxicity is far from understood. BMAA has agonistic properties for receptors like NMDA, AMPA/kainiteand metabotropic glutamate receptor 5 (mGluR5). It has been proposed that its main neurotoxicity mechanism is to be a receptor agonist of glutamate. It has also been shown that BMAA increase intracellular calcium levelsand also increased production of ROS. Later research has shown that BMAA induces abnormal tau-hyperphosphorylation in neurons and also inhibits the activity of protein phosphatase 2A (PP2A).

Our aim was to look further into the cellular effects BMAA both in vitro on neural cells and in vivo in zebrafish. By elucidating the mechanism of BMAA toxicity we will hopefully also obtain a broader understanding of how these neurodegenerative diseases are induced, which might open for new treatment targets.

We found that BMAA effect the cell viability of both SH-SY5Y neuroblastoma cell lines and C6 glioma cell lines. It also reduces the heartbeat in zebrafish larvae after exposure. We also saw that BMAA alters protein expression within cells and also in vivo zebrafish. Western blot analysis revealed that BMAA exposure increases the levels of GSK-3α/β in both neuroblastoma cells and glioma cells, as well as in zebrafish larvae. The antioxidant, oncogenic protein DJ-1 had also an increased expression after BMAA expression, while we observed a decrease in the expression of the antioxidant enzyme superoxide dismutase (SOD1).