Browsing by Subject "alpha-synuclein"

Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.

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.

Misfolding and aggregation of alpha-synuclein (α-syn) protein, leading to dysfunctional proteins and toxic protein aggregates, are seen as major factors in the pathogenesis of Parkinson’s disease (PD). Direct protein-protein interactions (PPI) between α-syn and a serine endopeptidase, prolyl oligopeptidase (PREP), have been shown to increase α-syn aggregation. Small molecular PREP inhibitors, in turn, have been shown to reduce the ɑ-syn aggregation process both in vitro and in vivo. Inhibition of PREP has been shown to have dual effects on ɑ-syn aggregation: first of all, blocking PREP mediated seeding and secondly, inducing the clearance of ɑ-syn aggregates via increased autophagy. Thus, PREP inhibitors should be further studied as a potential treatment for PD and other synucleinopathies. In this study, we evaluated the effect of two different PREP inhibitors, 4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047) and HUP-115 in a virus vector-based unilateral A53T-ɑ-syn overexpression mouse model. AAV-A53T-ɑ-syn injections used in this study caused a significant increase in oligomer-specific alpha-synuclein (ɑ-synO5) immunoreactivity and a mild dopaminergic neuron loss, together with mild motor deficits. Neither 2-week PREP inhibition with KYP-2047 or 4-week PREP inhibition with HUP-115 reduced ɑ-synO5 immunoreactivity or protected dopaminergic neurons in the substantia nigra (SN). Concordant to this, the treatments did not restore the slight behavioral deficit AAV-A53T-ɑ-syn injections caused in the cylinder test. In previous studies, PREP inhibition with KYP-2047 decreased ɑ-synO5 immunoreactivity, attenuated dopaminergic neuron loss and restored behavioral deficits in other α-syn overexpression mouse models. It is suggested that PREP inhibitors mainly have an effect on soluble ɑ-syn oligomers, rather than insoluble fibrils. In case A53T-ɑ-syn forms insoluble fibrils too rapidly in mice, overexpression of A53T-ɑ-syn might not be a suitable option when studying the effects of PREP inhibitors. Our results suggest that further characterization of this model in mice is much needed before drawing any conclusions about the effect of these PREP inhibitors.