Browsing by Subject "UPR"

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.

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.

IER3IP1 is a protein located in the endoplasmic reticulum (ER) transmembrane, and it is highly expressed in pancreatic beta cells and developing brain cortex. The loss-of-function mutations in IER3IP1 cause monogenic neonatal diabetes together with brain linked diseases such as epilepsy and microcephaly. The aim of this thesis is to study the role of IER3IP1 in the development and function of human beta cells using hESC-derived pancreatic islets. Using CRISPR/Cas9, IER3IP1 knockout (KO) and IER3IP1 loss-of-function mutation knock-in (KI) hESC clones were generated. For KO, the first exon of IER3IP1 was deleted whereas for KI, the 21. valine of IER3IP1 was changed to glycine. The clones together with their unedited controls (H1), were differentiated into pancreatic stem cell (SC)-islets following the optimized 7-stage differentiation protocol. The differentiation was followed during the protocol and the SC-islets were tested at the end of the protocol. In vitro, IER3IP1 KO-islets contained less beta cells and more alpha cells when compared to the H1-islets, as shown by immunostainings for insulin and glucagon. The beta cells of KO-islet accumulated more proinsulin compared to H1-islets and had significantly higher level of ER-stress shown by elevated ER-stress marker BiP. Moreover, the KO-islets showed drastically lower amount of insulin secretion and diminished insulin content. The IER3IP1 KI-islets did not significantly differ from H1-islets. Thus, this master’s thesis shows that IER3IP1 is essential for maintaining normal ER homeostasis and beta cell function in vitro. In future, these results should be confirmed using in vivo model.