It is estimated that about 10 million people are suffering from Parkinson's disease worldwide. As the prevalence of PD is increasing and the population is aging, the PD treatment market size is expected to grow and may reach 5.69 billion USD by 2022. There is a great unmet need in curative therapies and, thus, also in research aiming at a greater understanding of the disease-causing mechanisms.
Pathways protecting post-mitotic neurons from pathological protein aggregation
The presence of Lewy bodies – intraneuronal inclusions containing aggregated and phosphorylated α-synuclein as a main component – is a characteristic feature in the brains of both sporadic and familial Parkinson’s disease patients. Accumulation of phospho-α-synuclein-positive neuronal aggregates can compromise neuronal functions already at the early stage of PD by causing synaptic and mitochondrial dysfunction, cell stress, and deregulation of protein degradation pathways.
In collaboration with Prof. Kelvin Luk, Prof. Mart Saarma and Dr. Mikko Airavaara we developed a unique and original experimental system utilizing specifically defined conditions to model α-synuclein aggregation induced with pre-formed fibrils in cultured dopaminergic neurons and in aged wild-type mice and rats. We are currently further developing this system to use human induced pluripotent stem cells (iPSC)-derived dopaminergic neurons.
Combined with in-house-developed high content imaging and automated image analysis, our system gives a unique possibility to robustly and reliably follow the changes in α-synuclein phosphorylation in response to experimental drugs in dopaminergic neurons. Importantly, treatment with an experimental drug significantly attenuated α-synuclein phosphorylation in dopaminergic neurons. Using a combination of specific pathway inhibitors, CRISPR-Cas9-mediated gene editing and recombinant lentivirus-based transgenesis, we have identified molecular pathways that attenuate α-synuclein aggregation and promote neuronal survival.
Identification and characterization of neuroprotective microRNAs and compounds stimulating microRNA biogenesis
A double-stranded RNA-specific nuclease Dicer is a crucial enzyme in the biogenesis pathway of mature functional microRNAs. We have demonstrated that Dicer is essential for functions and survival of adult dopaminergic neurons, whereas its selective ablation leads to rapid and progressive loss of dopaminergic neurons and locomotor impairments. Our results suggest that an age-related and Parkinson’s disease-linked decline of Dicer expression results in deregulation of microRNA networks and compromises the function of aged dopaminergic neurons, contributing to their degeneration in Parkinson’s disease.
Dicer activity can be pharmacologically stimulated with enoxacin - a small molecule enhancing pre-microRNA cleavage in the Dicer protein complex. We have found that enoxacin treatment promoted survival of primary embryonic dopaminergic neurons. Our recent data indicate that enoxacin administration stimulates Dicer activity in vivo and rescues locomotor phenotype in mouse Parkinson’s disease model and that its neuroprotective effects require functional microRNA biogenesis.
We are currently elucidating molecular mechanisms of enoxacin action by microRNA expression profiling and pathway analysis, aiming to find neuroprotective microRNAs and their targets. We are also optimizing Dicer activity reporter assay for high throughput screening of chemical compound libraries to find small molecule drugs stimulating microRNA biogenesis. Such drugs can support the survival of dopaminergic neurons and may thus hold therapeutic potential for Parkinson’s disease treatment.
Cross-talk between α-synuclein and tau aggregation and uptake, ER stress, and protein degradation
Aggregated α-synuclein may accumulate in the endoplasmic reticulum (ER), activate unfolded protein response pathway and induce ER stress contributing to the degradation of dopaminergic neurons. By binding to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump α-synuclein aggregates can affect cytosolic and ER calcium levels. However, the effects of α-synuclein aggregates on ER function are not well studied. Aggregated α-synuclein impairs clathrin-mediated endocytosis (CME) and recycling of synaptic vesicles. CME is also one of the main routes of exogenous α-synuclein entrance to the cell. MicroRNAs may be important mediators of CME and, particularly, α-synuclein internalization. Moreover, pathophysiological data suggest a considerable cross talk between PD and Alzheimer’s disease, as α-synuclein can co-localize and physically interact with tau, disrupt tau-tubulin interactions and promote tau hyperphosphorylation leading to its aggregation.
Combining a multidisciplinary approach and collaboration network with expertise in molecular and cellular biology, transgenic animal models, recombinant virus-mediated transgenesis, pharmacology, electron and light microscopy, and high content image analysis, we study the effects of α-synuclein aggregation on ER morphology and calcium levels in primary mouse and human iPSC-derived dopaminergic neurons, and in different cellular compartments (neuronal soma and dendritic spines). We also study the impact of selected microRNAs on CME genes and α-synuclein internalization in primary dopaminergic neurons, and a cross-talk between α-synuclein and tau aggregation.
Multiparametric characterization of primary mouse and human iPSC-derived neurons
We have established a system utilizing high-content fluorescent microscopy imaging to analyze the effects of small molecules, neurotrophic factors, and microRNAs on survival and morphology of primary neuronal cultures. Utilizing primary midbrain cultures from mice with specific expression of green fluorescent protein (GFP) we can follow the morphology of GFP-positive dopaminergic neurons by live cell imaging. Moreover, using lentiviral vector-based transgenesis, fluorescent reporters and live cell imaging, we are currently establishing protocols to follow the activation of autophagy pathways and intracellular and ER Ca2+ levels in primary neuronal cultures and human iPSC-derived neurons. We employ multielectrode arrays to follow electrical activity in neuronal cultures and measure dopamine content in growth media with high-performance liquid chromatography.
In collaboration with Dr. Emilia Peltola (Aalto University), Prof. Tomi Taira and Dr. Anne Panhelainen (University of Helsinki) we aim to develop a multifunctional analysis platform with high sensitivity and a high-throughput format for long-term/multi time-point, simultaneous recordings of neurochemical activities (dopamine release) using fast-scan cyclic voltammetry method, recordings of electrical activities of the neurons, and high-content imaging. This platform will enable a detailed correlation of multiple neuronal readouts like never before, to screen the neuronal responses to pathological stressors relevant for Parkinson’s disease and to candidate neuroprotective drugs. We aim to demonstrate the applicability of our system for multiparametric analysis and quality control of human iPSC-derived dopaminergic neurons and for investigating the molecular mechanisms promoting dopaminergic neuron survival and functions.
In addition to a unique combination of tools and scientific expertise, we have established a strong collaboration network that includes leading national and international experts in the following fields:
- Neurotrophic factors and PD models: Prof. Mart Saarma, Prof. Raimo Tuominen, Dr. Mikko Airavaara (University of Helsinki)
- α-synuclein aggregation and spread: Prof. Kelvin Luk (University of Pennsylvania, USA); Prof. Maria Spillantini (University of Cambridge, UK)
- MicroRNA biology and expression profiling in Parkinson’s disease patients: Prof. Ilya Vinnikov (Shanghai Jiao Tong University, China); Prof. Kai C. Sonntag (Harvard Medical School, USA)
- DNA and RNA sequencing: Dr. Petri Auvinen (University of Helsinki)
- HPLC analysis of dopamine and its metabolites: Dr. Petteri Piepponen (University of Helsinki)
- Human iPSCs cultures and neuronal differentiation: Prof. Timo Otonkoski (University of Helsinki)
- Transgenic mouse models: Dr. Witold Konopka (Nencki Institute of Experimental Biology, Warsaw, Poland); Dr. Grzegorz Kreiner (Institute of Pharmacology, Krakow, Poland); Dr. Rosanna Parlato (Ulm University, Germany)
- Somatic transgenesis and single cell sequencing: Dr. Valery Grinevich (DKFZ Heidelberg, Germany); Prof. Konstantin Khodosevich (University of Copenhagen, Denmark)
- Development of reporter systems: Dr. Brandon Harvey (National Institute on Drug Abuse, USA)
- Targeted drug delivery using nanoparticles: Dr. Hélder A. Santos (University of Helsinki)
We also use core facilities in Viikki and Meilahti campuses: