Browsing by master's degree program "Master's Programme in Translational Medicine"

Renewal of the intestinal epithelium is driven by the actively dividing and strictly regulated Lgr5-expressing intestinal stem cells (ISCs). As uncontrolled proliferation may lead to colorectal cancer (CRC), ISCs and the regulatory circuit around them could elucidate new targets for colorectal cancer therapy. The regulatory crosstalk between the stem cells and surrounding stroma is under intensive investigation, but little is known about the neuronal control of the intestinal epithelium. Adrenergic signals from sympathetic nervous system regulate hematopoietic and melanocyte stem cells and promote tumorigenesis in e.g., pancreatic, and prostate cancer. Adra2a, one of the nine different adrenergic receptors, is expressed in the ISCs, and adrenergic neurons project neurites to the stem cell niche suggesting a paracrine signaling role. However, whether the adrenergic signaling plays a role in ISC regulation and/or in colorectal cancer remains unknown. The aim of this study was to develop tools to investigate the role of adrenergic signaling in ISC regulation. First, I set up a protocol to delete Adra2a and other genes in intestinal organoids recapitulating the stem cell hierarchy of the intestinal epithelium in vitro, using CRISPR-Cas9 technology. This led to successful deletion of the Ret proto-oncogene while an Adra2a-deficient organoid pool could not be established with these efforts. I differentiated neuroblastoma cells to a catecholaminergic phenotype and cultured them together with intestinal organoids to address the effect of catecholaminergic signaling on the intestinal epithelium in organoid cocultures. The coculture with catecholaminergic cells induced upregulation of the regeneration markers Ly6a and Clu in intestinal organoids, while Reg3b as well as the stem cell markers Lgr5, Olfm4, and Adra2a and the Paneth cell marker Lyz1 were reduced. Third, I assessed the direct effect of a 3-hour norepinephrine (NE) pulse on wild-type organoids with 3’RNA sequencing, however, this did not induce significant changes in the expression levels of the respective regeneration marker genes. Altogether, my work established a CRISPR-Cas9-based method to delete genes of interest in primary intestinal organoids. Further investigation is needed to verify if NE contributes to the regulation of intestinal regeneration and stem cell maintenance.

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.

Patients over 65 years are more prone to face difficulties in their care due to not only complex and chronic multimorbid conditions, but also fragmentation of health care services. Current healthcare systems are designed for single-disease conditions that do not align with the care needs of the multimorbid elderly. Multimorbid long-term home care clients use a wide range of home care services but also other health and social services outside of home care, which can lead to fragmentation. The study aimed to map out services used by multimorbid home care clients, present disruptions related to their care, and suggest feasible and scalable solutions for the identified disruptions. Home care professionals (N=10) and clients (N=5) were interviewed focusing on the services and disruptions, and a focus group workshop was conducted for health and elderly care specialists (N=9) for creating the solutions. A total of 38 individual disruptions were discovered, of which 58% (22 cases) mentioned more than one interviewee. The results indicate that multimorbid home care clients faced the most care disruptions when care was prescribed outside of home care and caused fragmentation in the care coordination and sharing of patient information between multiple care providers and actors. Other disruptions were caused by a lack of co-creation of health, inconsistencies within home care protocols, and other factors outside of home care such as rapid workforce turnover. The disruptions discovered were mainly related to healthcare service networks rather than social-related care, due to the healthcare services’ lack of care-related integration at multiple levels and dimensions which was not necessarily needed with social services. Possible solutions suggested by health and elderly care specialists included adapting current healthcare systems to the needs of the home care, improving care coordination through various means, utilizing digital solutions, creating tools to track the status of the client’s care, and increasing co-creation of health with the client. With the current challenges in recruiting and maintaining health care personnel in the home care and constant training of personnel; this study suggests that different types of technological solutions are needed to improve care coordination and integration.

Alzheimer’s disease (AD) is a neurodegenerative brain disorder in which the disease process may take decades until the symptoms become evident. To date, no ideal biomarker has emerged that would enable early detection of AD. Environmental and lifestyle factors are thought to affect the risk of developing AD, possibly through epigenetic mechanisms such as DNA methylation (DNAm). DNAm has been shown to differ in the blood and brain of subjects with AD compared with subjects without AD, suggesting that DNAm may be involved in the pathogenic process of AD. This study aims to detect the difference in blood DNAm at baseline between cases who later developed AD and controls who remained AD diagnosis-free during follow-up in a sample selected from a Finnish population-based cohort. Leucocyte genome-wide DNAm was profiled on approximately 850,000 CpG sites by using Infinium MethylationEPIC assay. Each CpG was regressed on the outcome of AD diagnosis during follow-up, controlling for subjects’ age at sampling, sex, smoking status, blood cell counts, working stress level, slide, and array. Specific differentially methylated positions (DMPs) were further explored using pathway analysis. Finally, the methylation level of the candidate gene (APOE) selected from the literature was compared with the sample of this study. After correction for multiple testing, the later diagnosis of AD was not significantly (adjusted p-value < 0.05) associated with methylation level at the baseline at any DNAm site. There was, however, a robust hypomethylation of DMPs among the cases, as 90.9% of the DMPs (p-value < 0.05) were hypomethylated in the case group. The 200 genes annotated by DMPs with the smallest p-values were involved in two neuronal pathways: “Axon guidance associated with semaphorins Homo sapiens” (p-value = 0.0058, adjusted p-value = 0.065) in Panther 2016 and “Semaphorin interactions Homo sapiens” (p-value = 0.00005, adjusted p-value = 0.078) in Reactome 2016. No significant difference existed in DNAm of the candidate gene (APOE) between cases and controls, while cg26190885 at the promoter region of APOE showed nominal significance (p-value = 0.04). In conclusion, no strong evidence was found to support the hypothesis that systemic changes in DNAm are involved in the pathogenesis of AD or that DNAm marks could be detected in blood before the symptoms become evident. A genome-wide pattern of hypomethylation measured by the Infinium MethylationEPIC assay was observed in the case group, serving as a venue for further investigations.

Mitochondrial aminoacyl tRNA-synthetases (mt-aaRS) catalyse the charging of tRNAs with their cognate amino acids in mitochondria. Mutations in mt-aaRS cause tissue-specific mitochondrial diseases, especially affecting tissues with high energy expenditure like the nervous system, heart, and kidneys. However, disease mechanisms for the heterogeneous group of diseases have not yet been fully elucidated. Harnessing CRISPR-Cas9 genome editing in induced pluripotent stem cells (iPSC) provides an opportunity to model mt-aaRS mutations in vitro and investigate the effects of individual mutations on cellular phenotype. SARS2 encodes mitochondrial seryl tRNA-synthetase, and its c.1347 G>A mutation causes severe childhood-onset progressive spastic paresis. Here, CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology directed repair (HDR) the genome of iPSC and knock-in the patient mutation. Guide RNAs were designed and tested for efficiency before electroporation into wild type iPSC. Clonal cell lines were made by low-density seeding and manual colony picking. The expression of pluripotency markers was measured by RT-qPCR. RT-qPCR and Western blot measured SARS2 mRNA expression and protein level respectively. The success and precision of genome editing were analysed by Sanger sequencing, comparing the performance of the different guide RNAs, and screening regions of potential off-target genome editing. Two genome-edited iPSC lines with the SARS2 c.1347 G>A mutation were successfully generated to model the patient mutation. The iPSC lines expressed pluripotency markers and contained no off-target genome editing and modelled the patient’s decrease in SARS2 protein level and mRNA expression. More evidence of differentiation ability is needed before differentiation into the affected cell type (motor neurons) and further disease modelling. The efficiency of CRISPR-Cas9 for genome editing, especially harnessing HDR in iPSC, is an area of future research.

Distal myopathies are a group of inherited diseases that cause progressive muscle weakness primarily in the hands and feet. Variants in various different genes have been identified as disease-causing, but most recently, variants in ACTN2, a gene previously associated with cardiomyopathy, have been shown to cause a distal myopathy phenotype. ACTN2 encodes alpha-actinin-2, an important structural protein that links actin and titin to the sarcomere Z-disk. In ACTN2-related diseases, actininopathies, several variants have been identified as disease-causing, however, new variants are continuously discovered, and the significance of many variants remain unknown. Thus, lack of clear genotype-phenotype correlations in actininopathies persists. Further, the molecular mechanisms underlying actininopathies are largely unknown, especially in recessive actininopathies. Here, a reanalysis of the previously reported ACTN2-related molecular and clinical findings is conducted, with a subsequent reclassification of variants according to American College of Medical Genetics and Genomics (ACMG) guidelines. The results indicate that ACTN2 serves an important function in the muscle tissue and is involved in the pathomechanisms of myopathy, which is supported by a growing body of clinical, genetic, and functional evidence. However, distinct genotype-phenotype correlations are currently clearly understood only in some actininopathies, and limited segregation and functional data are still available to support the pathogenicity of most previously reported missense variants. Additionally, functional characterization of ACTN2 variants identified in recessive actininopathies suggest that the underlying molecular mechanisms of recessive actininopathies are different form the dominant from of the disease, as they do not produce detectable alpha-actinin-2 aggregates in the cell models. Thus, alternate methods should be used to investigate the molecular mechanisms of recessive actininopathies. Also, the results suggest that multiple different molecular mechanisms are involved in dominant actininopathies.

Antiepileptic drugs (AED) are vital for treating epilepsy, but their use in pregnancy carries significant risks. Prenatal exposure to some AEDs like valproic acid increases the risk of teratogenicity and deficits in cognitive development in children. The impact of AEDs at different ages has been studied but their effect on the trajectory of cognitive development remains unknown. This study assessed the cognitive performance of children with prenatal AED exposure and unexposed controls using Bayley scales of infant and toddler development III (BSID-III) at 2 years and Weschler intelligence scales for children (WISC-IV) at 6 years of age. The association between 2- and 6-year outcomes was analyzed with separate ANCOVA models for each WISC-IV subscale. BSID-III subscales were used as the covariates and the models were adjusted for confounding factors. BSID-III scores in cognitive (B=6.70, p=0.01) and language (B=6.92, p=0.008) subscales were significantly associated with the WISC-IV working memory scores in controls but not in the exposed group. The groups were also found to differ in their intercorrelations between BSID-III subscale scores. Scores at 2 years were not associated with later results in exposed children as they were in controls. This may suggest that the cognitive development trajectory of AED-exposed children differs from that of unexposed controls, possibly following an alternate projection. Developmental trajectories should be considered when investigating the cognitive effects of prenatal AED exposure. The finding of lacking correlations of BSID-III subscales also raises cause for future investigation of the structure of cognition in AED exposed children.

Objectives. Ageing is accompanied by neurobiological changes, such as changes in grey matter (GM) volume and cortical thickness, that mediate a gradual cognitive decline, which can, in turn, be potentially offset by stimulating leisure activities. Choir singing is an especially feasible musical activity with positive effects on physiological, psychological, cognitive, and social functioning in old age. Research investigating the effects of choir singing on the ageing brain is limited. As part of the Brain, Ageing, and Vocal Expression (BRAVE) project, this study aimed to investigate the effect of ageing and choir singing on GM structure. Methods. Using a cross-sectional design and voxel-based morphometry (VBM) and surface-based morphometry (SBM), this study compared GM structure between young (20-39 years; n=35), middle-aged (40-59 years; n=34), and old (60-90 years; n=31) participants and investigated the interaction of age and choir singing on GM structure with amateur choir singer (n=54) and controls (n=46). Results and conclusions. Age had a significant and widespread effect on GM structure, with old participants showing lower GM volume and cortical thickness than young (in bilateral sensorimotor, auditory/language, visual, and limbic areas, midbrain, and cerebellum) and middle-aged (in right visual cortex, thalamus, hippocampus and left auditory cortex) participants. Middle-aged participants also showed lower GM volume and cortical thickness than young participants (in bilateral sensorimotor, language, and visual areas, basal ganglia, cerebellum, and right hippocampus and amygdala). These results corroborate the current understanding of neurobiological ageing. No significant interaction of age and choir singing was found on GM structure, which could be explained by methodological factors. Further research is needed to determine whether choir singing can support brain structure or function across healthy ageing.

In this master’s thesis, in vitro neuromuscular junction (NMJ) model was set up using microfluidic devices. Additionally, the effect of R878H/R878H mutation in MCM3AP gene that causes an early-onset peripheral neuropathy on NMJ formation and maintenance was studied. To study human NMJs that significantly differ from other mammal NMJs is challenging and new models to study the function of these complex and highly specialized structures are needed. Induced pluripotent stem cells (iPSC) and motor neurons were characterized with gene expression studies using qRT-PCR and with immunocytochemistry studies using commonly known markers for pluripotency and motor neurons. NMJs were studied in 2D co-cultures and with microfluidic devices. Gene expression studies were conducted from 2D co-cultures and co-cultures in microfluidic devices provided detailed information of the localization and morphology of NMJs. Expression of essential genes for NMJ formation together with immunocytochemistry results with alpha-bungarotoxin (BTX) staining showed that NMJs were formed in both control and R878H/R878H mutant cell line co-cultures. There was a trend of lower gene expression levels of NMJ essential genes in the R878H/R878H mutant line compared to the control line and also immunocytochemistry results indicated impairment in NMJ formation in the mutant line, but further studies are needed to validate the effect of R878H/R878H mutation on the NMJ formation. In future, functional studies could be conducted to investigate whether these NMJs are functional and the information from the motor neuron terminal is conveyed to the muscle membrane.

The contact site between the endoplasmic reticulum and mitochondria, also known as the mitochondria endoplasmic reticulum contact sites (MERCS), have a crucial role in maintaining the homeostasis within the cell. Across the MERCS multiple functions, such as regulation of calcium (Ca2+) homeostasis, lipid metabolism, ER stress, mitochondrial quality control (MQC) and regulation of unfolded protein response (UPR) take place. These processes have been shown to be implicated in numerous different neurodegenerative diseases, such as Parkinson’s disease. Parkinson’s disease is the second most common neurodegenerative disease that at the moment has no cure. The main obstacle in developing a neuroprotective treatment for the disease is the limited understanding of the key molecular events leading to neurodegeneration. One of the things in Parkinson’s disease that has eluded scientists for years is the selective death of the dopaminergic (DA) neurons in substantia nigra pars compacta. One hypothesis that could explain the selective death is the Ca2+ hypothesis, looking at the Ca2+ vulnerability of SNpc DA neurons as a plausible cause leading to the selective cell death. This project focused looking at the protein level and morphological changes of the ER and MERCS in stressed neurons, hypothesizing these as possible sites that contribute to the neuron vulnerability, as they are known to be the key modulators of the intracellular Ca2+ homeostasis. This study looked closer at two MERC proteins, GRP75 and BAP31, and one ER protein, SERCA2, to see how they are affected in stressed dopamine-like neurons. Firstly, the in vitro model was established by differentiating SH-SY5Y neuroblastoma cells to dopamine-like neurons expressing tyrosine hydroxylase. Three different molecular compounds were tested as possible stressors affecting the Ca2+ homeostasis within the neurons, and we concluded that thapsigargin, a commonly used stressor to model PD like pathology, leads to the highest measurable ER Ca2+ depletion. Lastly, we quantitatively and qualitatively analyzed the effect of 24-hour treatment with each stressor on the differentiated SH-SY5Y neurons. Thapsigargin treatment lead to an increased level of GRP75 and SERCA2. A slight increase in BAP31 was also detected after thapsigargin treatment, but no apparent changes of the ER morphology were detected. The results, together with previous research, show GRP75 to be a possible contributor to the pathology of the disease, but further research is needed to see if it could be a possible target for treatment.