Browsing by Subject "alpha-synuclein"

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  • Pätsi, Henri T.; Kilpeläinen, Tommi P.; Auno, Samuli; Dillemuth, Pyry M. J.; Arja, Khaled; Lahtela-Kakkonen, Maija K.; Myöhänen, Timo T.; Wallen, Erik A. A. (2021)
    Different five-membered nitrogen-containing heteroaromatics in the position of the typical electrophilic group in prolyl oligopeptidase (PREP) inhibitors were investigated and compared to tetrazole. The 2-imidazoles were highly potent inhibitors of the proteolytic activity. The binding mode for the basic imidazole was studied by molecular docking as it was expected to differ from the acidic tetrazole. A new putative noncovalent binding mode with an interaction to His680 was found for the 2-imidazoles. Inhibition of the proteolytic activity did not correlate with the modulating effect on protein-protein-interaction-derived functions of PREP (i.e., dimerization of alpha-synuclein and autophagy). Among the highly potent PREP inhibiting 2-imidazoles, only one was also a potent modulator of PREP-catalyzed alpha-synuclein dimerization, indicating that the linker length on the opposite side of the molecule from the five-membered heteroaromatic is critical for the disconnected structure-activity relationships.
  • Albert, Katrina; Voutilainen, Merja H.; Domanskyi, Andrii; Airavaara, Mikko (2017)
    Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV--synuclein (-syn) to target substantia nigra dopamine neurons to produce an -syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.
  • Airavaara, Mikko; Parkkinen, Ilmari; Konovalova, Julia; Albert, Katrina; Chmielarz, Piotr; Domanskyi, Andrii (2020)
    Abstract Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. ? 2020 The Authors.
  • Lindholm, Dan; Pham, Dan D.; Cascone, Annunziata; Eriksson, Ove; Wennerberg, Krister; Saarma, Mart (2016)
    Parkinson's disease (PD) is a progressive neurodegenerative disorder causing movement disabilities and several non-motor symptoms in afflicted patients. Recent studies in animal models of PD and analyses of brain specimen from PD patients revealed an increase in the level and activity of the non-receptor tyrosine kinase Abelson (c-Abl) in dopaminergic neurons with phosphorylation of protein substrates, such as alpha-synuclein and the E3 ubiquitin ligase, Parkin. Most significantly inhibition of c-Abl kinase activity by small molecular compounds used in the clinic to treat human leukemia have shown promising neuroprotective effects in cell and animal models of PD. This has raised hope that similar beneficial outcome may also be observed in the treatment of PD patients by using c-Abl inhibitors. Here we highlight the background for the current optimism, reviewing c-Abl and its relationship to pathophysiological pathways prevailing in PD, as well as discussing issues related to the pharmacology and safety of current c-Abl inhibitors. Clearly more rigorously controlled and well-designed trials are needed before the c-Abl inhibitors can be used in the neuroclinic to possibly benefit an increasing number of PD patients.
  • Hlushchuk, Irena; Barut, Justyna; Airavaara, Mikko; Luk, Kelvin; Domanskyi, Andrii; Chmielarz, Piotr (2022)
    There are several links between insulin resistance and neurodegenerative disorders such as Parkinson's disease. However, the direct influence of insulin signaling on abnormal alpha-synuclein accumulation-a hallmark of Parkinson's disease-remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological alpha-synuclein accumulation induced by the addition of oc-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect alpha-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on alpha-synuclein accumulation. We observed, however, a reduced alpha-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of alpha-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced oc-synuclein phosphorylation in a primary dopaminergic cell culture.
  • Reunanen, Saku (Helsingin yliopisto, 2020)
    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.
  • Lehtonen, Sarka; Sonninen, Tuuli-Maria; Wojciechowski, Sara; Goldsteins, Gundars; Koistinaho, Jari (2019)
    Despite decades of research, current therapeutic interventions for Parkinson's disease (PD) are insufficient as they fail to modify disease progression by ameliorating the underlying pathology. Cellular proteostasis (protein homeostasis) is an essential factor in maintaining a persistent environment for neuronal activity. Proteostasis is ensured by mechanisms including regulation of protein translation, chaperone-assisted protein folding and protein degradation pathways. It is generally accepted that deficits in proteostasis are linked to various neurodegenerative diseases including PD. While the proteasome fails to degrade large protein aggregates, particularly alpha-synuclein (alpha-SYN) in PD, drug-induced activation of autophagy can efficiently remove aggregates and prevent degeneration of dopaminergic (DA) neurons. Therefore, maintenance of these mechanisms is essential to preserve all cellular functions relying on a correctly folded proteome. The correlations between endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) that aims to restore proteostasis within the secretory pathway are well-established. However, while mild insults increase the activity of chaperones, prolonged cell stress, or insufficient adaptive response causes cell death. Modulating the activity of molecular chaperones, such as protein disulfide isomerase which assists refolding and contributes to the removal of unfolded proteins, and their associated pathways may offer a new approach for disease-modifying treatment. Here, we summarize some of the key concepts and emerging ideas on the relation of protein aggregation and imbalanced proteostasis with an emphasis on PD as our area of main expertise. Furthermore, we discuss recent insights into the strategies for reducing the toxic effects of protein unfolding in PD by targeting the ER UPR pathway.
  • Singh, Abhishek (Helsingin yliopisto, 2019)
    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.
  • Chmielarz, Piotr; Er, Safak; Konovalova, Julia; Bandres, Laura; Hlushchuk, Irena; Albert, Katrina; Panhelainen, Anne; Luk, Kelvin; Airavaara, Mikko; Domanskyi, Andrii (2020)
    Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder Society
  • Oinas, Minna; Polvikoski, Tuomo; Sulkava, Raimo; Myllykangas, Liisa; Juva, Kati; Notkola, Irma-Leena; Rastas, Sari; Niinisto, Leena; Kalimo, Hannu; Paetau, Anders (2009)
  • Cui, Hengjing; Norrbacka, Susanna Maria; Myöhänen, Timo (2022)
    The accumulation of aggregated alpha-synuclein (alpha-syn) has been identified as the primary component of Lewy bodies that are the pathological hallmarks of Parkinson's disease (PD). Several preclinical studies have shown alpha-syn aggregation, and particularly the intermediates formed during the aggregation process to be toxic to cells. Current PD treatments only provide symptomatic relief, and alpha-syn serves as a promising target to develop a disease-modifying therapy for PD. Our previous studies have revealed that a small-molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, increases alpha-syn degradation by accelerating macroautophagy (MA) leading to disease-modifying effects in preclinical PD models. However, alpha-syn is also degraded by chaperone-mediated autophagy (CMA). In the present study, we tested the effects of PREP inhibition or deletion on CMA activa-tion and alpha-syn degradation. HEK-293 cells were transfected with alpha-syn and incubated with 1 & 10 mu M KYP-2047 for 24 h. Both 1 & 10 mu M KYP-2047 increased LAMP-2A levels, induced alpha-syn degradation and reduced the expression of Hsc70, suggesting that the PREP inhibitor prevented alpha-syn aggregation by activating the CMA pathway. Similarly, KYP-2047 increased the LAMP-2A immunoreactivity and reduced the Hsc70 levels in mouse primary cortical neurons. When LAMP-2A was silenced by a siRNA, KYP-2047 increased the LC3BII/LC3BI ratio and accelerated the clearance of alpha-syn. Additionally, KYP-2047 induced CMA effectively also when MA was blocked by bafilomycin A1. Based on our results, we suggest that PREP might function as a core network node in MA-CMA crosstalk, and PREP inhibition can reduce alpha-syn levels via both main autophagy systems.
  • Julku, Ulrika H.; Jäntti, Maria; Svarcbahs, Reinis; Myöhänen, Timo T. (2021)
    Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial effects in Parkinson's disease models by enhancing the clearance of aSyn aggregates and modulating striatal dopamine. Additionally, we have shown that PREP regulates phosphorylation and internalization of dopamine transporter (DAT) in mice. In this study, we clarified the mechanism behind this by using HEK-293 and PREP knock-out HEK-293 cells with DAT transfection. We tested the effects of PREP, PREP inhibition, and alpha-synuclein on PREP-related DAT regulation by using Western blot analysis and a dopamine uptake assay, and characterized the impact of PREP on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) by using PKC assay and Western blot, respectively, as these kinases regulate DAT phosphorylation. Our results confirmed our previous findings that a lack of PREP can increase phosphorylation and internalization of DAT and decrease uptake of dopamine. PREP inhibition had a variable impact on phosphorylation of ERK dependent on the metabolic state of cells, but did not have an effect on phosphorylation or function of DAT. PREP modifications did not affect PKC activity either. Additionally, a lack of PREP elevated a DAT oligomerization that is associated with intracellular trafficking of DAT. Our results suggest that PREP-mediated phosphorylation, oligomerization, and internalization of DAT is not dependent on PKC or ERK.
  • Er, Safak; Hlushchuk, Irena; Airavaara, Mikko; Chmielarz, Piotr; Domanskyi, Andrii (2020)
    The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson’s disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.
  • Ignatius, Adele (Helsingin yliopisto, 2021)
    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.
  • Ahola, Sari (Helsingfors universitet, 2015)