Browsing by Subject "ER stress"

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  • Valkonen, Konsta Valentin (Helsingin yliopisto, 2021)
    Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motoneuron disease. ALS is characterized by a progressive loss of upper and lower motoneurons, resulting in muscle atrophy, paralysis and ultimately in death. Approximately 30,000 people die of ALS annually. There is no cure for ALS, and only two drugs - riluzole and edavarone - have been approved for the treatment of the disease. The complex pathology of ALS contributes to the lack of effective treatments. Several cellular pathologies have been suggested to contribute to the pathogenesis, including ER stress, disruption of calcium homeostasis, oxidative stress and excitotoxicity. Here we describe the cytoprotective effects of C-terminal fragments of the novel proteins with neurotrophic factor properties MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) on a toxin model of ALS in vitro. Unlike the classical neurotrophic factors, MANF and CDNF are predominantly localized to the endoplasmic reticulum (ER) and have been shown to alleviate ER stress by keeping the unfolded protein response (UPR) transducers inactive. ER stress is a major component in many neurodegenerative diseases, including ALS, and is a promising therapeutic target for MANF and CDNF. However, the potential of these proteins in ALS treatment remains to be insufficiently described. We used differentiated motoneuron-like NSC-34 cells treated with a range of toxins, modelling different cellular pathologies linked to ALS. After the toxin addition, we treated the cells with MANF and CDNF variants and riluzole and measured the cell viability. The toxin panel consists of tunicamycin, ionomycin and staurosporine. Tunicamycin causes cell death by activating proapoptotic branches of the UPR. Ionomycin is an ionophore and depletes the ER of calcium, thus inducing both UPR-dependent and UPR-independent apoptosis. Less is known about the mechanisms of staurosporine, but it has been shown to induce caspase-3-dependent apoptosis, increase intracellular calcium levels and cause oxidative stress. We hypothesized that both MANF and CDNF variants protect the cells against UPR-dependent apoptosis but not against UPR-independent cell death. We show that MANF and CDNF variants protect the cells against apoptosis induced by tunicamycin, ionomycin and staurosporine. Interestingly, the protein variants mediated the highest protection against ionomycin-induced stress, and they exhibited mild protective effects against staurosporine as well. These findings suggest that MANF and CDNF variants might have a role in maintaining intracellular calcium homeostasis. However, it is possible that staurosporine induces ER stress as well, which would explain the protection conferred by the protein variant. We report that the CDNF variant mediates higher protection at lower concentrations compared to the MANF variant in every toxin assay, whereas the MANF variant mediates higher protection at the highest tested concentration compared to the CDNF variant. We also show that the CDNF variant-mediated protection against staurosporine-induced stress peaked at lower concentrations, and the highest concentration provided distinctively lower, yet significant effect. These data lead us to hypothesize that the protein variants may have a slightly different mode of action, and that they might provide an additive effect when administered simultaneously. We tested a combination of MANF and CDNF variants in cells treated with tunicamycin, ionomycin and staurosporine. However, the combination treatment did not increase the viability more than MANF and CDNF variants independently did. The results answered our questions as well as raised new ones. In the future, the putative calcium-regulating effects of the protein variants should be investigated. The UPR-modifying effects of the drug candidates and toxins need to be assessed by quantifying changes in the UPR marker mRNA and protein expression levels. If it is revealed that the variants have a different mode of action, the possible additive protective effects must be assessed. Finally, a wider toxin panel is needed to fully explore the potential of MANF and CDNF variants in ALS treatment. This study demonstrates the potential of MANF and CDNF variants in protecting motoneurons against several pathological pathways contributing to ALS pathology. However, the mechanisms of action of the variants need further investigation to fully understood their therapeutic potential.
  • Pakarinen, Emmi; Lindholm, Paivi; Saarma, Mart; Lindahl, Maria (2022)
    Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) display cytoprotective effects in animal models of neurodegenerative diseases. These endoplasmic reticulum (ER)-resident proteins belong to the same protein family and function as ER stress regulators. The relationship between CDNF and MANF function, as well as their capability for functional compensation, is unknown. We aimed to investigate these questions by generating mice lacking both CDNF and MANF. Results showed that CDNF-deficient Manf(-/-) mice presented the same phenotypes of growth defect and diabetes as Manf(-/-) mice. In the muscle, CDNF deficiency resulted in increased activation of unfolded protein response (UPR), which was aggravated when MANF was ablated. In the brain, the combined loss of CDNF and MANF did not exacerbate UPR activation caused by the loss of MANF alone. Consequently, CDNF and MANF deficiency in the brain did not cause degeneration of dopamine neurons. In conclusion, CDNF and MANF present functional redundancy in the muscle, but not in the other tissues examined here. Thus, they regulate the UPR in a tissue-specific manner.
  • Almeida, Sérgio (Helsingfors universitet, 2016)
    Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) form a novel neurotrophic factor family due to their unique structure and different mode of action when compared to classical neurotrophic factors. CDNF and MANF have shown to protect dopaminergic neurons in Parkinson's disease animal models and therefore they are considered potential therapy agents. However, their target molecules, i.e., putative receptor(s) and signalling pathways are still unknown. 78 kDa glucose-regulated protein (GRP78) member of the heat shock protein (HSP) family is a major chaperone that under Endoplasmic Reticulum (ER) stress conditions is up-regulated and prevents protein aggregation as well as facilitates degradation of misfolded proteins. It locates mainly in the ER but location can change in different conditions. In cancer research, GRP78 has been found highly expressed on the surface of cancer cells where it regulates critical oncogenic signalling pathways. For example, it was recently shown that Par-4 (Prostate apoptosis response-4) induces apoptosis via activation of caspase-3 by binding to GRP78, expressed at the surface of cancer cells. GRP78 has been shown capable of relocating extracellularly also in neurons. Especially, it was recently shown that accumulating extracellular α-synuclein induces an increase in surface-exposed GRP78 in cultured neurons. α-synuclein interacts with cell surface GRP78 and activates a signalling cascade affecting the morphology and dynamics of actin cytoskeleton. Our group has recent, yet unpublished data suggesting that CDNF and MANF interact with GRP78 protein. The emerging role for GRP78 also in the neurodegeneration requests further investigation on its possible interaction with CDNF and MANF and on the biological meaning of that interaction. In order to test whether CDNF and MANF would interact with cell surface GRP78 and possibly compete with par-4 for the binding and in this way prevent apoptosis, we built a plasmid that would guide the expression and extracellular localization of GRP78 in the transfected cells. We transfected HEK293 cells with this plasmid and incubated them for 24h with two concentrations of par-4. We could see a trend of increasing apoptosis in PAR-4 –treated cells, but this was not enhanced in the cells expressing GRP78 extracellularly, as we had hypothesised. Thus we did not continue further with testing CDNF and MANF on this setting. Transfected HEK293 cells were incubated with alkaline phosphatase tagged MANF or CDNF (AP-MANF or AP-CDNF) and using the alkaline phosphatase substrate pNitrophenylphosphate (pNPP), we were able to study the binding between GRP78 and CDNF and MANF. Even though we could not prove the cell surface GRP78 interaction with MANF with this method, we show a high affinity binding between cell surface GRP78 and CDNF when transfected cells are incubated with different concentrations of AP-CDNF.
  • Lantto, Tiina A.; Laakso, Into; Dorman, H. J. Damien; Mauriala, Timo; Hiltunen, Raimo; Köks, Sulev; Raasmaja, Atso (2016)
    Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.
  • Wang, Liang; Li, Menglu; Bu, Qian; Li, Hongchun; Xu, Wei; Liu, Chunqi; Gu, Hui; Zhang, Jiamei; Wan, Xuemei; Zhao, Yinglan; Cen, Xiaobo (2019)
    Much efforts have been tried to clarify the molecular mechanism of alcohol-induced brain damage from the perspective of genome and protein; however, the effect of chronic alcohol exposure on global lipid profiling of brain is unclear. In the present study, by using Q-TOF/MS-based lipidomics approach, we investigated the comprehensive lipidome profiling of brain from the rats orally administrated with alcohol daily, continuously for one year. Through systematically analysis of all lipids in prefrontal cortex (PFC) and striatum region, we found that long-term alcohol exposure profoundly modified brain lipidome profiling. Notably, three kinds of lipid classes, glycerophospholipid (GP), glycerolipid (GL) and fatty acyls (FA), were significantly increased in these two brain regions. Interestingly, most of the modified lipids were involved in synthetic pathways of endoplasmic reticulum (ER), which may result in ER stress-related metabolic disruption. Moreover, alcohol-modified lipid species displayed long length of carbon chain with high degree of unsaturation. Taken together, our results firstly present that chronic alcohol exposure markedly modifies brain lipidomic profiling, which may activate ER stress and eventually result in neurotoxicity. These findings provide a new insight into the mechanism of alcohol-related brain damage.
  • Sket, Tina (Helsingin yliopisto, 2020)
    Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which leads to the activation of unfolded protein response (UPR) through three transmembrane protein sensors located in the ER membrane. The sensors correspond to three branches of the UPR, namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) branches. Upon ER stress, IRE1 dimerizes and oligomerizes, and its endonuclease domain is activated. It specifically targets X-box-binding protein 1 (XBP1) mRNA, from which a 26 nt intron is spliced. This allows a complete translation of spliced XBP1 mRNA into a functional protein that acts as a transcription factor. Together with the other pathways, the UPR leads to a decrease in the protein folding load by causing a reduction in the general level of protein translation, and by inducing the expression of protein folding machinery. However, if the UPR is activated continuously for a long time, the apoptotic pathway will be triggered, and the cell will die. ER stress and UPR are associated with various disorders, such as some types of cancer, diabetes, chronic inflammatory syndromes, and particularly neurodegeneration. For example, in Parkinson’s disease, it was suggested that prolonged ER stress induces the extensive apoptosis of dopaminergic neurons in substantia nigra pars compacta region of the midbrain. This hinders the normal functioning of the nigrostriatal pathway, and hence results in the progressive development of Parkinson’s motor symptoms. In order to study the regulation or IRE1 branch of the UPR, and to identify the ER-stress-modulating compounds, a human luciferase reporter cell line (XBP1-NLuc) was created in this work. The reporter was expressed when IRE1 splicing was activated, since the XBP1 intron fragment was fused to the Nano luciferase gene. The expression of the reporter was observed with luciferase assay at several time points during treatments. The treatments were done with ER stress inducers thapsigargin and tunicamycin, and with IRE1 inhibitors KIRA6 and 4μ8c, or the combination of those. Quantitative PCR (qPCR) was used to validate the expression of the reporter and to monitor the expression of the other branches of the UPR. Additionally, the oligomerization of IRE1 was observed with IRE1-GFP cell line that was treated identically to the XBP1-NLuc cell line, fixed, stained for nuclei, and imaged with fluorescent microscopy. After imaging, the IRE1-GFP clusters were analysed and quantified with CellProfiller and CellAnalyst softwares. Both cell lines were used to test the effect of neurotrophic factors CDNF, MANF, and MANF mutant isomers on the UPR with and without tunicamycin treatment. Collectively, the experiments confirmed that XBP1-NLuc cell line was created successfully and that it accurately reports IRE1 splicing activity. As expected, ER stress treatment increased the reporter expression, while IRE1 inhibitors decreased the expression of the reporter. qPCR revealed that the other observed UPR markers were activated as well upon thapsigargin treatment, however, they were not decreased with the treatment with IRE1 specific inhibitors. In line with XBP1-NLuc cell line, the IRE1-GFP cell line demonstrated an increased oligomerization of IRE1 upon ER stress induction. The KIRA6 inhibitor of IRE1, which prevents IRE1 oligomerization, decreased the formation of IRE1-GFP clusters. Additionally, the IRE1-endonuclease-activity inhibitor 4μ8c induced the formation of IRE1-GFP clusters. Curiously, the distribution of the intensity of IRE1-GFP clusters was bimodal and could point to two manners of IRE1 clustering and/or activation. Together, the experiments done with cells transfected with CDNF, MANF or MANF mutants, suggested that the tested neurotrophic factors decreased IRE1 oligomerization and its activation. However, there were substantial problems in the quantification of viable cells, which should be considered in the interpretation of these results. No significant difference among the tested neurotrophic factors was observed. In conclusion, the XBP1-NLuc reporter cell line provided a reliable reporter of IRE1 endonuclease activity, whose expression is increased during the ER stress. Together with IRE1-GFP cell line, it revealed the amount of IRE1 oligomerization and activation under various treatments and at different time points relative to treatments. Due to the effectiveness and accuracy, the XBP1-NLuc cell line can be further used in studying the regulation and activation of IRE1, as well as for the identification of ER-stress modulating molecules, which can be used for development of novel treatments for ER stress associated diseases, such as Parkinson’s disease.
  • Lindholm, Dan; Mäkelä, Johanna; Di Liberto, Valentina; Mudo, Giuseppa; Belluardo, Natale; Eriksson-Rosenberg, Ove; Saarma, Mart (2016)
    Parkinson's disease (PD is a progressive neurological disorder characterized by the degeneration and death of midbrain dopamine and non-dopamine neurons in the brain leading to motor dysfunctions and other symptoms, which seriously influence the quality of life of PD patients. The drug L-dopa can alleviate the motor symptoms in PD, but so far there are no rational therapies targeting the underlying neurodegenerative processes. Despite intensive research, the molecular mechanisms causing neuronal loss are not fully understood which has hampered the development of new drugs and disease-modifying therapies. Neurotrophic factors are by virtue of their survival promoting activities attract candidates to counteract and possibly halt cell degeneration in PD. In particular, studies employing glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NRTN), as well as the recently described cerebral dopamine neurotrophic factor (CDNF) and the mesencephalic astrocyte-derived neurotrophic factor (MANF) have shown positive results in protecting and repairing dopaminergic neurons in various models of PD. Other substances with trophic actions in dopaminergic neurons include neuropeptides and small compounds that target different pathways impaired in PD, such as increased cell stress, protein handling defects, dysfunctional mitochondria and neuroinflammation. In this review, we will highlight the recent developments in this field with a focus on trophic factors and substances having the potential to beneficially influence the viability and functions of dopaminergic neurons as shown in preclinical or in animal models of PD.
  • Granqvist, Riikka (Helsingin yliopisto, 2021)
    Parkinson´s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer´s disease. There is still no drug that alters the state of the disease. It has been found that Endoplasmic reticulum (ER) stress is one mechanism in PD. ER stress occurs due to accumulation of unfolded proteins. ER stress triggers Unfolded protein response (UPR) that protects against ER stress by decreasing unfolding of proteins. In the beginning, UPR has protective effect, but in prolonged ER stress UPR triggers apoptotic cell death. There are several key mediators in the UPR pathway. Characterisation of ER stress in PD models may be important for the current and future drug development of PD. If ER stress is a significant factor that affects the disease development, it would be important to find a drug that alters these mechanisms and UPR. This may be a way to halt the disease development. Different animal models of PD, like 6-OHDA (6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model, have similarities in their mechanisms. It has been found that ER stress occurs both in the brain of PD patients and animal models of PD. That is why studying and further characterisation in animal models is relevant. The aim of this study was to characterize ER stress in 6-OHDA rat model. The expression of some key mediators of the UPR were determined in this study. There were male and female Spraque Dawley rats in this experiment. 6-OHDA or saline was injected intrastriatally in 3 spots by stereotaxic surgery. Two weeks after 6-OHDA lesions, amphetamine-induced rotation test was conducted to the rats. The rats were divided into groups based on lesion size according to the results. For this study, the rats were euthanised at week 2 or week 4 post lesion. The rats were euthanised by carbondioxide, and the death was confirmed by decapitation. The brains were collected and stored in -80°C. Striatum and substantia nigra were collected later. Total RNA was isolated from these samples. Part of the RNA sample was used to conduct cDNA synthesis. Finally, the gene expression of Atf4, Ire1α, Xbp1s, Xbp1t, Grp78 and Chop was measured from these cDNA samples by qPCR (quantitative polymerase chain reaction). The qPCR data describes the expression of exact gene. The data was processed prior to statistical analysis. By statistical analysis, it was possible to compare the expression of these genes between 6-OHDA group and vehicle group. In addition, comparison was made between 6-OHDA treated groups at week 2 and 4. According to the results, only Chop expression had increased in 6-OHDA lesioned rats at week 2 compared to the vehicle group. In other genes there were no statistical differences, unlike in several other studies where the expression was found to be increased. Thus, the characterisation of this model requires further studying, possibly by increasing the sample size and studying later time points as well.
  • Srinivasan, Vignesh; Bruelle, Celine; Scifo, Enzo; Pham, Dan Duc; Soliymani, Rabah; Lalowski, Maciej; Lindholm, Dan Bj (2020)
    USP14 is a deubiquitinating enzyme associated with the proteasome that is important for protein degradation. Here we show that upon proteasomal inhibition or expression of the mutant W58A38 USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70 in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, but also to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whilst inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or the use of mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis by various drugs may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways
  • Lehtonen, Sarka; Sonninen, Tuuli-Maria; Wojciechowski, Sara; Goldsteins, Gundars; Koistinaho, Jari (2019)
    Despite decades of research, current therapeutic interventions for Parkinson's disease (PD) are insufficient as they fail to modify disease progression by ameliorating the underlying pathology. Cellular proteostasis (protein homeostasis) is an essential factor in maintaining a persistent environment for neuronal activity. Proteostasis is ensured by mechanisms including regulation of protein translation, chaperone-assisted protein folding and protein degradation pathways. It is generally accepted that deficits in proteostasis are linked to various neurodegenerative diseases including PD. While the proteasome fails to degrade large protein aggregates, particularly alpha-synuclein (alpha-SYN) in PD, drug-induced activation of autophagy can efficiently remove aggregates and prevent degeneration of dopaminergic (DA) neurons. Therefore, maintenance of these mechanisms is essential to preserve all cellular functions relying on a correctly folded proteome. The correlations between endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) that aims to restore proteostasis within the secretory pathway are well-established. However, while mild insults increase the activity of chaperones, prolonged cell stress, or insufficient adaptive response causes cell death. Modulating the activity of molecular chaperones, such as protein disulfide isomerase which assists refolding and contributes to the removal of unfolded proteins, and their associated pathways may offer a new approach for disease-modifying treatment. Here, we summarize some of the key concepts and emerging ideas on the relation of protein aggregation and imbalanced proteostasis with an emphasis on PD as our area of main expertise. Furthermore, we discuss recent insights into the strategies for reducing the toxic effects of protein unfolding in PD by targeting the ER UPR pathway.
  • Hulmi, Juha J.; Hentila, Jaakko; DeRuisseau, Keith C.; Oliveira, Bernardo M.; Papaioannou, Konstantinos G.; Autio, Reija; Kujala, Urho M.; Ritvos, Olli; Kainulainen, Heikki; Korkmaz, Ayhan; Atalay, Mustafa (2016)
    Protein homeostasis in cells, proteostasis, is maintained through several integrated processes and pathways and its dysregulation may mediate pathology in many diseases including Duchenne muscular dystrophy (DMD). Oxidative stress, heat shock proteins, endoplasmic reticulum (ER) stress and its response, i.e. unfolded protein response (UPR), play key roles in proteostasis but their involvement in the pathology of DMD are largely unknown. Moreover, exercise and activin receptor IIB blocking are two strategies that may be beneficial to DMD muscle, but studies to examine their effects on these proteostasis pathways are lacking. Therefore, these pathways were examined in the muscle of mdx mice, a model of DMD, under basal conditions and in response to seven weeks of voluntary exercise and/or activin receptor IIB ligand blocking using soluble activin receptor-Fc (sAcvR2B-Fc) administration. In conjunction with reduced muscle strength, mdx muscle displayed greater levels of UPR/ER-pathway indicators including greater protein levels of IREloc, PERK and Atf6b mRNA. Downstream to IREloc and PERK, spliced Xbpl mRNA and phosphorylation of elF2oc, were also increased. Most of the cytoplasmic and ER chaperones and mitochondrial UPR markers were unchanged in mdx muscle. Oxidized glutathione was greater in mdx and was associated with increases in lysine acetylated proteome and phosphorylated sirtuin 1. Exercise increased oxidative stress when performed independently or combined with sAcvR2B-Fc administration. Although neither exercise nor sAcvR2B-Fc administration imparted a clear effect on ER stress/UPR pathways or heat shock proteins, sAcvR2B-Fc administration increased protein expression levels of GRP78/BiP, a triggering factor for ER stress/UPR activation and TxNIP, a redox-regulator of ER stress-induced inflammation. In conclusion, the ER stress and UPR are increased in mdx muscle. However, these processes are not distinctly improved by voluntary exercise or blocking activin receptor IIB ligands and thus do not appear to be optimal therapeutic choices for improving proteostasis in DMD. (C) 2016 Elsevier Inc. All rights reserved.
  • Voutilainen, Merja H.; De Lorenzo, Francesca; Stepanova, Polina; Bäck, Susanne; Pulkkila, Päivi; Pörsti, Eeva; Saarma, Mart; Männistö, Pekka T.; Tuominen, Raimo K. (2017)
    Parkinson's disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic (DAergic) neurons of the substantia nigra (SN) and the accumulation of intracellular inclusions containing alpha-synuclein. Current therapies do not stop the progression of the disease, and the efficacy of these treatments wanes over time. Neurotrophic factors (NTFs) are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. CDNF (cerebral dopamine NTF) and GDNF (glial cell line-derived NTF) are able to protect DAergic neurons against toxin-induced degeneration in experimental models of PD. Here, we report an additive neurorestorative effect of coadministration of CDNF and GDNF in the unilateral 6-hydroxydopamine (6-OHDA) lesion model of PD in rats. NTFs were given into the striatum four weeks after unilateral intrastriatal injection of 6-OHDA (20 mu g). Amphetamine-induced (2.5 mg/kg, i.p.) rotational behavior was measured every two weeks. Number of tyrosine hydroxylase (TH)-positive cells from SN pars compacta (SNpc) and density of TH-positive fibers in the striatum were analyzed at 12 weeks after lesion. CDNF and GDNF alone restored the DAergic function, and one specific dose combination had an additive effect: CDNF (2.5 mu g) and GDNF (1 mu g) coadministration led to a stronger trophic effect relative to either of the single treatments alone. The additive effect may indicate different mechanism of action for the NTFs. Indeed, both NTFs activated the survival promoting PI3 kinase (PI3K)-Akt signaling pathway, but only CDNF decreased the expression level of tested endoplasmatic reticulum (ER) stress markers ATF6, glucose-regulated protein 78 (GRP78), and phosphorylation of eukaryotic initiation factor 2 alpha subunit (eIF2 alpha).
  • Taha, Lamia (Helsingin yliopisto, 2021)
    The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.
  • Pakarinen, Emmi; Danilova, Tatiana; Voikar, Vootele; Chmielarz, Piotr; Piepponen, Petteri; Airavaara, Mikko; Saarma, Mart; Lindahl, Maria (2020)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) localized protein that regulates ER homeostasis and unfolded protein response (UPR). The biology of endogenous MANF in the mammalian brain is unknown and therefore we studied the brain phenotype of MANF-deficient female and male mice at different ages focusing on the midbrain dopamine system and cortical neurons. We show that a lack of MANF from the brain led to the chronic activation of UPR by upregulation of the endoribonuclease activity of the inositol-requiring enzyme 1 alpha (IRE1 alpha) pathway. Furthermore, in the aged MANF-deficient mouse brain in addition the protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6) branches of the UPR pathways were activated. Neuronal loss in neurodegenerative diseases has been associated with chronic ER stress. In our mouse model, increased UPR activation did not lead to neuronal cell loss in the substantia nigra (SN), decrease of striatal dopamine or behavioral changes of MANF-deficient mice. However, cortical neurons lacking MANF were more vulnerable to chemical induction of additional ER stress in vitro. We conclude that embryonic neuronal deletion of MANF does not cause the loss of midbrain dopamine neurons in mice. However, endogenous MANF is needed for maintenance of neuronal ER homeostasis both in vivo and in vitro.
  • Koppinen, Tapani Kalle (Helsingin yliopisto, 2019)
    Multiple sclerosis (MS) is a demyelinating autoimmune disease in which peripheral immune cells infiltrate the CNS and damage the insulating myelin sheaths surrounding neurons, creating demyelinated lesions in the spinal cord and the brain. MS is an incurable, life-long disease which causes a range of symptoms resulting from CNS degeneration. Current treatments mostly focus on preventing autoimmune attacks and the formation of lesions, but do not reduce the damage caused by the attacks, or impact the gradual degeneration of the axons of MS patients. This study aimed to establish the potential of MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) as treatments for MS. MANF and CDNF are endoplasmic reticulum (ER) located proteins with unique structure and mode of action. UPR is a cellular stress response that, when triggered by inflammation in MS, can cause the apoptosis of myelinating oligodendrocytes and neurodegeneration. MANF and CDNF are also capable of modulating immune responses and improving regenerative processes in damaged tissues. The capability of these two molecules to protect CNS tissue was tested on mice induced with experimental autoimmune encephalomyelitis (EAE), a disease model for MS. Intravenous injections of MANF or CDNF in two doses were performed every 2nd day for 28 days after disease induction. Behavioral testing (rotarod and open field tests) indicated that both proteins improved motor function before the onset of paralysis. Daily clinical scoring showed a brief therapeutic window after the onset of paralysis, during which MANF and CDNF were able to halt disease progression. Flow cytometry analysis of mice spleens and brains showed no effect on immune cell populations at the end of the 28-day testing period. Immunohistological staining at the end of the experiment showed no differences in levels of neuroinflammation between treatment groups and control mice but showed that treatment with MANF and CDNF clearly reduced the formation of demyelinated lesions over the duration of the disease. These findings suggest the improved motor performances and protection from paralysis provided by treatment by MANF and CDNF may be due to their ability to protect CNS tissue from UPR caused by autoimmune demyelinating attacks. Further research is required to elucidate the mechanics behind this neuroprotective ability, and lead to more effective use of MANF and CDNF.
  • Lankinen, Tuuli (Helsingin yliopisto, 2020)
    Our hearing perception is based on the ability to discriminate mechanical sound waves and to amplify and transduce them into electrical stimuli.This function is based on the complex cellular organization of the cochlea, the hearing organ. The sensory epithelium in the organ of Corti spirals along the cochlear duct in a tonotopic arrangement: every sound frequency elicits the strongest response at allocation along this duct. Sound stimulus is detected by three rows of outer hair cells (OHCs) which amplify- and tone-discriminate the sound stimulus, and by one row of inner hair cells (IHCs), which transduce the mechanical stimulus into electric impulses. Basal regions of the cochlea detect high- frequency sounds and apical regions detect low- frequency sounds. The complexity and sensitivity of the cochlea is linked with its vulnerability to various traumas. Most kinds of damage to the mammalian hair cells is irreversible, because these cells are not capable of regeneration. Hearing impairment has many etiologies. Common to them is that damage is permanent and no pharmacotherapy is available. Hearing impairment is often a disabling condition and it has vast societal consequences. The number of hearing impaired people is constantly increasing and the WHO has estimated that 10% of the world`s population will suffer from disabling hearing loss in 2050. Mesencephalic astrocyte- derived neurotrophic factor (MANF) is an unconventional, ER-resident protein that promotes ER- homeostasis. It has been associated with cytoprotective functions in many neurodegenerative disease- models and shown to promote recovery after ischemic trauma. MANF expression has been previously found in many cell-types in the cochlea, including OHCs and IHCs. Its deficiency in a mouse model led to upregulation of ER-stress markers and a robust, tonotopic base –to apex gradient loss of outer hair cells and severe hearing loss. This study examines the role of MANF in noise-induced trauma in the hair cells of the cochlea. In a conditionally inactivated (Manf -/- cKO) mouse model in the C57BL/6J – background, where Manf has been inactivated from most of the cochlear cells, I studied, if Manf -deficiency sensitizes the cells to noise-induced cell death in two age-groups. I also examined the basic and noise- induced MANF expression, using two mouse- strains, C57BL/6J and CBA/Ca. I also examined OHC stereociliary bundle morphology to find out if noise induces morphological changes in Manf cKO-mice that differ from noise-exposed C57BL/6j wild type mice. This study found that OHCs have a low MANF- expression, whereas in IHCs the expression is strong. MANF is expressed in a base- to apex gradient in the OHCs of the two mouse-strains examined, in a uniform pattern, that correlates with vulnerability, implicating that low levels of MANF predispose basal OHCs to vulnerability. MANF expression in the IHCs was non-gradiental. Noise did not induce upregulation, as was expected, but instead noise induced downregulation of MANF in the basal region of the OHCs by an unknown mechanism in both mouse-strains.This suggests that noise-induced trauma induces ER dyshomeostasis, possibly independent of ER stress response pathways ,unfold protein response (UPR). This study also demonstrates that MANF deficiency sensitizes the OHCs to noise- induced trauma, resulting in more elevated OHC loss and hearing thresholds. This sensitization is mainly caused by a progressive degenerative changes seen in the OHC stereociliary bundles of Manf cKO-mice, and is associated with more severe noise-induced hearing loss. The results of my study suggest that MANF has an important, yet unknown, protective role in noise-induced trauma in OHCs. These results support the possible role of MANF as a therapeutic agent in a noise-induced trauma.
  • Järvinen, Elli Katariina (Helsingin yliopisto, 2021)
    Ischemic stroke is a complex disease involving multiple pathophysiological mechanisms. To date, many therapeutic intervention strategies such as anti-inflammatory treatments have been tested, but none of them has been successful. Previous studies have shown that mesencephalic astrocyte-derived neurotrophic factor (MANF) improves stroke recovery and increases the expression of phagocytosis related genes. In this study, the phagocytic and inflammatory effect of monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), complement component 3 (C3), adhesion G protein-coupled receptor E1 (ADGRE1), MER receptor tyrosine kinase (MerTK) and mesencephalic astrocyte-derived neurotrophic factor (MANF) on microglia were studied simultaneously for the first time. The phagocytosis related genes were transiently transfected into a microglial cell line and studied in vitro utilizing phagocytosis assay, fluorescence-activated cell sorting, Western blot and enzyme-linked immunosorbent assay. MCP-1, M-CSF and C3a were shown to enhance microglial phagocytosis without inducing a pro-inflammatory response. In addition, MerTK induces phagocytosis and the synthesis of pro-inflammatory cytokines. In conclusion, the real therapeutic potential of MCP-1, M-CSF, C3a and MerTK in stroke treatment should be further characterized and tested in vivo.
  • Makinen, Selina; Nguyen, Yen H.; Skrobuk, Paulina; Koistinen, Heikki A. (2017)
    Saturated fatty acids are implicated in the development of insulin resistance, whereas unsaturated fatty acids may have a protective effect on metabolism. We tested in primary human myotubes if insulin resistance induced by saturated fatty acid palmitate can be ameliorated by concomitant exposure to unsaturated fatty acid oleate. Primary human myotubes were pretreated with palmitate, oleate or their combination for 12 h. Glucose uptake was determined by intracellular accumulation of [H-3]-2-deoxy-d-glucose, insulin signalling and activation of endoplasmic reticulum (ER) stress by Western blotting, and mitochondrial reactive oxygen species (ROS) production by fluorescent dye MitoSOX. Exposure of primary human myotubes to palmitate impaired insulin-stimulated Akt-Ser(473), AS160 and GSK-3 beta phosphorylation, induced ER stress signalling target PERK and stress kinase JNK 54 kDa isoform. These effects were virtually abolished by concomitant exposure of palmitate-treated myotubes to oleate. However, an exposure to palmitate, oleate or their combination reduced insulin-stimulated glucose uptake. This was associated with increased mitochondrial ROS production in palmitate-treated myotubes co-incubated with oleate, and was alleviated by antioxidants MitoTempo and Tempol. Thus, metabolic and intracellular signalling events diverge in myotubes treated with palmitate and oleate. Exposure of human myotubes to excess fatty acids increases ROS production and induces insulin resistance.
  • Sonninen, Tuuli-Maria; Goldsteins, Gundars; Laham-Karam, Nihay; Koistinaho, Jari; Lehtonen, Sarka (2020)
    Protein homeostasis (proteostasis) disturbances and inflammation are evident in normal aging and some age-related neurodegenerative diseases. While the proteostasis network maintains the integrity of intracellular and extracellular functional proteins, inflammation is a biological response to harmful stimuli. Cellular stress conditions can cause protein damage, thus exacerbating protein misfolding and leading to an eventual overload of the degradation system. The regulation of proteostasis network is particularly important in postmitotic neurons due to their limited regenerative capacity. Therefore, maintaining balanced protein synthesis, handling unfolding, refolding, and degrading misfolded proteins are essential to preserve all cellular functions in the central nervous sysytem. Failing proteostasis may trigger inflammatory responses in glial cells, and the consequent release of inflammatory mediators may lead to disturbances in proteostasis. Here, we review the mechanisms of proteostasis and inflammatory response, emphasizing their role in the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, we discuss the interplay between proteostatic stress and excessive immune response that activates inflammation and leads to dysfunctional proteostasis.
  • Hubel, Einav; Fishman, Sigal; Holopainen, Minna; Käkelä, Reijo; Shaffer, Ortal; Houri, Inbal; Zvibel, Isabel; Shibolet, Oren (2021)
    Drug-induced liver injury is an emerging form of acute and chronic liver disease that may manifest as fatty liver. Amiodarone (AMD), a widely used antiarrhythmic drug, can cause hepatic injury and steatosis by a variety of mechanisms, not all completely understood. We hypothesized that repetitive AMD administration may induce hepatic lipotoxicity not only via effects on the liver but also via effects on adipose tissue. Indeed, repetitive AMD administration induced endoplasmic reticulum (ER) stress in both liver and adipose tissue. In adipose tissue, AMD reduced lipogenesis and increased lipolysis. Moreover, AMD treatment induced ER stress and ER stress-dependent lipolysis in 3T3L1 adipocytes in vitro. In the liver, AMD caused increased expression of genes encoding proteins involved in fatty acid (FA) uptake and transfer (Cd36, Fabp1, and Fabp4), and resulted in increased hepatic accumulation of free FAs, but not of triacylglycerols. In line with this, there was increased expression of hepatic de novo FA synthesis genes. However, AMD significantly reduced the expression of the desaturase Scd1 and elongase Elovl6, detected at mRNA and protein levels. Accordingly, the FA profile of hepatic total lipids revealed increased accumulation of palmitate, an SCD1 and ELOVL6 substrate, and reduced levels of palmitoleate and cis-vaccenate, products of the enzymes. In addition, AMDtreated mice displayed increased hepatic apoptosis. The studies show that repetitive AMD induces ER stress and aggravates lipolysis in adipose tissue while inducing a lipotoxic hepatic lipid environment, suggesting that AMD-induced liver damage is due to compound insult to liver and adipose tissue. NEW & NOTEWORTHY AMD chronic administration induces hepatic lipid accumulation by several mechanisms, including induction of hepatic ER stress, impairment of b-oxidation, and inhibition of triacylglycerol secretion. Our study shows that repetitive AMD treatment induces not only hepatic ER stress but also adipose tissue ER stress and lipolysis and hepatic accumulation of free fatty acids and enrichment of palmitate in the total lipids. Understanding the toxicity mechanisms of AMD would help devise ways to limit liver damage.