Browsing by Subject "MANF"

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.

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.

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.

Neurotrophic factors (NTFs) play an important role in regulating the survival, differentiation and maturation of developing neurons. Based on strong pre-clinical evidences, some of NTFs have been suggested to be efficient therapeutic agents for treatment of Parkinson’s disease (PD). PD is a neurodegenerative disorder characterized by loss of dopamine (DA) neurons from nigrostriatal pathway resulting in motor symptoms of the disease. A hallmark of the disease is the presence of Lewy bodies in the brain and they comprise majorly of aggregated alpha-synuclein (aSyn) protein. MANF, an unconventional NTF, was discovered over a decade ago and differs from traditional NTFs. Removal of MANF has been shown to trigger unfolded protein response in cells. Evidences indicate that increased endogenous level of aSyn may have a role in enhancing the process of aggregation of aSyn into Lewy body. Determining the initiation event of aSyn aggregation is an important step in Lewy body pathology and it is still under investigation. In the first part of this study, I aimed to elucidate if MANF knockout can trigger any change in endogenous level of aSyn. Transmission of Lewy bodies from cell to cell has been well studied by researchers and is suggested to spread across brain in a prion like fashion. CDNF has been neuroprotective and restorative for tyrosine hydroxylase (TH)-positive neurons in a toxin-based models of PD. However, presently exists no study which has evaluated the effects of CDNF on propagation of aSyn aggregates in vivo. In the second part of this study, I aimed at evaluating effects of long-term intrastriatal infusion of CDNF at two concentrations (1.5 μg/24h or 3 μg/24h) on propagation of endogenous phosphorylated aSyn inclusions in vivo. CRISPR/Cas9-mediated MANF knockout in SH-SY5Y cells did not yield any significant changes in the endogenous level of aSyn. Additionally, brain samples derived from MANF knockout mice yielded similar non-significant difference in level of aSyn compared to wild-type mice. MANF knockout primary DA neurons when inoculated either with only pre-formed fibrils (PFFs) or with a combination of PFFs and aSyn overexpression, showed no significant difference in the number of Lewy body like aggregates, suggesting no change in endogenous aSyn levels. Rats were injected with PFFs and then chronically infused with CDNF, 1 month and 2 months after PFFs at 2 different concentrations (1.5 μg/24h or 3 μg/24h). Immunohistochemical analysis of substantia nigra pars compacta (SNpc) derived from rats showed similar numbers of endogenous phosphorylated aSyn inclusions in animals treated chronically with either CDNF or PBS. In summary, only MANF knockout from cells or animals has no direct effect on endogenous level of aSyn. But external stressors may perhaps trigger upregulation of aSyn in MANF knockout cells. Furthermore, chronic infusion of CDNF either 1 month or 2 months after PFF injection doesn’t reduce the total number of phosphorylated aSyn inclusions in SNpc compared to control. Nevertheless, we need more data to corroborate this evidence.

Parkinson’s disease (PD) is a progressive chronic neurodegenerative disorder, which results in the selective loss of dopaminergic neurons in the substantia nigra (SN). The loss of these neurons results in the dysfunction of the nigrostriatal pathway bringing forth the characteristic motor symptoms seen in PD: postural instability, rigidity, slowness of movement and resting tremors. Non-motor symptoms, such as cognitive deficits, depression and impaired olfaction, typically emerge before motor symptoms. Currently available treatments only provide symptomatic relief with diminishing returns over time and no improvements on the overall outcome of the disease. Neurotrophic factors (NTF) have been of particular interest as a possible curative treatment for PD due to their potential for neuroprotection and neurorestoration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an NTF that has shown promising results in numerous in vitro and in vivo studies of PD. However, therapy with MANF and other NTFs involves surgical intervention for local administration, as NTFs cannot cross the blood-brain barrier (BBB). Therefore, the therapeutic potential of a systemically administered NTF would be tremendous, as it would lead to a significantly more favorable risk-benefit ratio for the patient. The aim of the current investigation is to evaluate the efficacy of a next generation variant of MANF in the 6-hydroxydopamine toxin-induced unilateral lesion rat model of PD. Prior in vivo results suggested that subcutaneously injected MANF variant is able to penetrate the BBB. Amphetamine-induced rotational behavior (AMPH-ROTO) was used to evaluate the severity of the unilateral lesions during the experiment every other week until the end of the experiment at week eight. Animals were divided into treatment groups during week two based on their AMPH-ROTO results. Animals received MANF variant either subcutaneously through an implanted osmotic minipump at two different dosages or as a single dose divided into three separate intrastriatal injections. Tyrosine hydroxylase (TH) immunohistochemical staining was performed on brain sections collected from the striatum and SN for data analysis. In addition to AMPH-ROTO results, the efficacy of treatment was determined via the optical density of TH-positive striatal fibers and the number of TH-positive cells in the SN. Statistically significant differences (defined by p < 0.05 and a non-zero mean difference at a 95 % confidence interval) were observed only in the number of TH-positive cells in the SN favoring intrastriatal MANF variant treatment over both intrastriatal MANF and the vehicle treatment. The main concern regarding the validity of the results was related to the heterogeneous lesion sizes in different treatment groups possibly resulting in unsuccessful randomization due to excessive baseline differences. The inadvertent negative effects of this was further exacerbated by low a priori statistical power, which in the end had likely caused inflated effect sizes. Thus, assessment of the definitions of the used statistical parameters and the limitations of the experimental design suggest that presently, the efficacy of the MANF variant could not be evaluated reliably, in spite of the statistically significant result.

Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.

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.

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.

Introduction: Oxidative stress occurs in cells when reactive oxygen species are generated as a by-product of oxygen metabolism and start to accumulate excessively. While extensive oxidative stress is highly detrimental to the cells, trophic factors help them survive. Trophic factor MANF has interested especially Parkinson’s disease researchers, but recent findings suggest that MANF plays a role in many diseases, also ones with an early childhood-onset. For this reason, it is important to investigate MANF function in different cell types. We have studied how MANF-knockout human embryonic stem cells react to oxidative stress compared to wild-type human embryonic stem cells, by exposing the cells to hydrogen peroxide and ethanol. Results: MANF-knockout human embryonic stem cells were more sensitive to oxidative stress than wild-type cells, but the variation between measurements was remarkable and the differences were statistically insignificant. We found that a transcription factor of our interest localized in the cell nuclei of MANF-knockout cells upon oxidative stress exposure. Such a nuclear translocation did not occur in wild-type cells. Moreover, we found that high concentrations (>2%) of ethanol reduced the viability of cells in only four hours. Discussion: Our findings suggest that MANF-knockout human embryonic stem cells react to oxidative stress differently than wild-type cells. Additional studies are necessary to clarify whether MANF-knockout human embryonic stem cells are indeed more sensitive to oxidative stress than wild-type cells. In the future, it would be interesting to inspect whether MANF protects human embryonic stem cells when the cells are exposed to physiologically relevant ethanol concentrations for longer periods of time.

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.