Browsing by Subject "α-synuclein"

Prolyl oligopeptidase (PREP) is a serine protease that is widely found throughout the human body and especially in the brain. The primary function of PREP is thought to be the hydrolysis of the carboxyl side bond of proline residues in oligopeptides. PREP is also shown to increase the dimerization and aggregation of α-synuclein and downregulate the protein phosphatase 2A mediated autophagy in the cell via direct protein-protein interactions (PPI). The accumulation of α-synuclein aggregates in cell is known to cause α-synucleinopathies such as Parkinson’s disease. This makes the PPIs of PREP an attractive target for drug research. The mechanisms of the PPIs of PREP are still poorly understood. Recent studies have shown that these PPIs can be modulated with ligands lacking high inhibitory activity for the proteolytic activity. These studies show that the IC50-value of the ligand does not correlate with ligands ability to affect the PPIs of PREP. Ligands that could selectively modulate the PPIs of PREP without inhibiting the proteolytic activity of PREP could give valuable information on the mechanisms of the PPIs and on how to modulate them. It is hypothesized that the ligands could bind to PREP at a site that does not interfere with its proteolytic activity, and ligand binding is assumed to restrict the dynamic structure of PREP and thereby also modulating the PPIs of PREP. The aim of this study was to synthetize novel peptidic PREP ligands and study their effects on the proteolytic activity of PREP and the PPIs of PREP. The aim was to find and identify ligands and structures that would modulate the PPIs of PREP and observe how the IC50-values of the ligands would correlate. L-Alanyl-pyrrolidine was selected as the scaffold for the compound series and the five-membered heteroaromatics, imidazole, triazole and tetrazole, were added to the 2-position of the pyrrolidine ring. In this position there is an electrophilic group in many PREP inhibitors, although these heteroaromatics are not electrophiles. The scaffold was also expanded by adding phenyalkyl groups with different linker lengths were added to the N-terminal side of the alanine. The ligands were synthesized using four synthesis routes which were based on synthesis methods found in literature. The IC50-values and the effects on α-synuclein dimerization and autophagic flux were determined for five synthetized compounds. The tested compounds were all weak PREP inhibitors and showed no strong activity in the α-synuclein dimerization and autophagy assays. Despite the weak activities in the assays, the importance of the linker length in the phenyalkyl group was shown. Changing the linker by one methylene group had noticeable effect on the overall activity. The results also demonstrate a lack of correlation between the IC50-values and the effects on α-synuclein dimerization and autophagic flux, which further confirms the lack of correlation between the proteolytic function and the PPIs of PREP which was observed also in previous studies.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with the neuropathological hallmark of intraneuronal inclusions called Lewy bodies (LB). Accumulation of α-synuclein (α-syn) and cellular components into LBs coincides with degeneration of dopaminergic neurons in the midbrain, substantia nigra. Degeneration of dopaminergic neurons eventually leads to motor dysfunctions. Currently, the treatments for PD are symptomatic. For this reason, new disease-modifying treatments are needed to slow down or prevent the disease progression. Neurotrophic factors (NTFs) have been an interest of research for a couple of decades because of their neuroprotective properties. The main aim of this study was to investigate if brain-derived neurotrophic factor (BDNF) reduces pre-formed fibril (PFF) induced aggregation of α-syn in dopaminergic neurons. PFF-model was used to mimic the accumulation of LBs in neurons, as PFFs induce aggregation of endogenous α-syn in neurons. Additionally, the dose dependence of BDNF was tested. The secondary objective was to investigate the interaction of tropomyosin receptor kinase B (TrkB) signaling pathway and α-syn aggregation using TrkB agonists and antagonists. The cultured dopaminergic neurons isolated from the midbrain of mouse embryos were treated with PFFs on the day in vitro (DIV) 8. BDNF or control treatments were added either 1 hour after the PFF-treatment or on DIV 12. Neurons were fixed on DIV 15 and fluorescent immunohistochemistry was performed. After the detection of fluorescence with automated, high-content imaging, image analysis was done for quantifying dopaminergic neurons, and dopaminergic neurons positive for LB-like aggregates by using unbiased image analysis CellProfilerTM software. Both BDNF and positive control glial cell line-derived neurotrophic factor (GDNF) significantly reduced LB-like aggregates in dopaminergic neurons at both timepoints. GDNF was more effective at both timepoints than BDNF. Both tested doses of BDNF lowered the number of LB-like aggregates, but a more robust effect was seen with the higher dose. The highest tested dose for the TrkB agonists was toxic to the cultured dopaminergic neurons, whereas the lower doses did not affect either the survival or the number of LB-like aggregates. BDNF promoted the survival of the dopaminergic neurons despite the survival-reducing adverse effect of TrkB antagonist K252a. This study provided new information on the effects of exogenously added BDNF on PFF-model with primary neuronal culture. Research on the underlying mechanisms of α-syn aggregation and the protective effects of NTFs can forward the development of new therapies against PD.