Neurodegenerative disorders are associated in a proportion of cases with genetic risk and gene mutations. However, the vast majority of cases of Parkinson’s disease are sporadic and the disease is altogether heterogeneous in symptoms and pathology. The Fitzgerald group aims to understand the molecular mechanisms underlying neurodegeneration using genetic forms of the disease as an entry point and a focus on the role of mitochondria.
Mitochondria are crucial organelles that produce energy and perform many other functions needed for central metabolism and cell signalling. Neurons use a lot of energy and rely on tight control for efficiency and to reduce oxidative burden. Mitochondrial dysfunction is a phenomenon that traverses all neurodegenerative diseases and may be an important in explaining the selective vulnerability of certain brain cells. This is especially relevant in Parkinson’s disease, where the dopaminergic neurons die, because the mitochondria are a major source of oxidative stress and the location of dopamine degradation.
In Parkinson’s disease, the link between mitochondrial dysfunction and disease has been proven by the identification of environmental factors and disease genes which critically affect mitochondria. The outcome has been a large body of work depicting the role of mitochondrial dysfunction in Parkinson’s disease, yet the exact mechanisms underlying sporadic forms of PD are less defined.
We utilize several different model cell systems with focus on patient-derived cells. In close collaboration with the Hertie Institute biobank and the Neurology Clinics, we are currently working in primary human fibroblasts derived from patients and healthy individuals, induced pluripotent stem cells (iPSCs), small molecule neuronal progenitor cells (smNPCs) and mature dopaminergic neurons. We are also working with peripheral blood cells (PBMCs) from patients and healthy individuals.
We are performing mostly functional work focusing on biology, biochemistry and physiology. We have established several specialized techniques for monitoring mitochondrial health and have also recently established several protein biochemistry, imaging and flow cytometry methods for investigating the endosomal-lysosomal system.
The role of PINK1 in an iPSC-derived neuronal model of Parkinson’s disease
Anna Schaedler, Christine Bus (Alumni)
We are currently investigating the role of the Parkinson’s disease kinase PINK1 in human dopaminergic neurons derived from iPSCs and using gene editing technology to generate isogenic controls. With several established readouts to assess mitochondrial function following different stress conditions, we were able to identify distinct mitochondrial phenotypes, which suggest a role for PINK1 in mitochondrial quality control. Using these PINK1 deficient models and their isogenic controls we will now look for genetic and pharmacological modifiers to apply novel neuroprotective strategies.
Mutations in the mitochondrial PTEN-induced putative kinase 1 (PINK1) are the second most common cause of recessive inherited Parkinson´s disease. The mechanism by which the loss of function mutation of PINK1 leads to the death of dopaminergic neurons in the mid brain remains elusive. Working with an iPSC derived model of dopaminergic neurons deficient in PINK1, we aim to gain further insights into the underlying mechanisms. In PINK1 knock out dopamine neurons no classical mitophagy after mitochondrial membrane depolarization was observed, while levels of autophagy and ROS production remained unchanged. The goal of the project is to find potential mechanisms of mitochondrial quality control/recycling in PINK1 knock out dopamine neurons in vitro.
The role of Miro1 in mitochondrial quality control
Mitochondria play a crucial role in several cellular mechanisms including energy supply, calcium homeostasis and apoptosis. While the matrix and mitochondrial inner membrane are well characterized, little is known about the mitochondrial outer membrane (MOM). The German Research Council research training group (RTG) MOMbrane aims to close this gap by to investigating the various functions of the MOM as well as its structure, regulation and biogenesis. The MOMbrane includes ten PhD students in different institutes in Tübingen, each focusing on a different aspect of the MOM. The project is carried out in collaboration with partner labs of the Weizmann institute in Rehovot (Israel).
Miro1 is located at the MOM and known to be responsible for mitochondrial transport. As part of the MOMbrane RTG, we aim to elucidate the function of Miro1 in mitochondrial turnover, metabolism, calcium signaling. It is known that one of the pathological hallmarks of Parkinson’s disease is altered mitochondrial function. We aim to better understand the biological roles of Miro1 and also its relevance to Parkinson’s disease by studying relevant disease models. To achieve this knowledge, we are introducing disease-associated and functional Miro1 variants in Miro1 in human induced pluripotent stem cells (iPSCs) using gene editing. Healthy and edited iPSCs can then be differentiated into other cell types including neurons.
The role of the endosomal-lysosomal system in atypical Parkinson’s disease/Corticobasal syndrome
Katharina Stegen (Alumni)
Dysfunction of the endosomal-lysosomal system is a phenomenon that traverses many neurodegenerative diseases including Alzheimer’s diseases, Parkinson’s disease and other rarer neurodegenerative diseases such as progressive supranuclear palsy (PSP) and corticobasal syndrome/degeneration (CBS/CBD). The endosomal lysosomal system is formed and driven by changes in pH and therefore the ion exchangers that reside in the membranous compartments of the endosomal lumen are critical for correct pH regulation. These exchangers are so crucial that major defects in them often cause severe mental retardation from birth. We are investigating defects that instead confer susceptibility to develop neurodegenerative diseases in later life. We are also investigating aspects of cellular trafficking involving the endosomal-lysosomal system and autophagy that could be involved in sporadic Parkinson’s disease.
Mitochondrial biomarkers in sporadic Parkinson’s disease
Julia Fitzgerald, Gerrit Machetanz, Anne Grünewald (LCSB, Université du Luxembourg)
Sporadic forms of Parkinson’s disease are the most common but they are difficult to model in the laboratory because there is no known gene mutation that can be introduced or corrected to generate isogenic controls. Statistically, large cohorts are needed to compare healthy individuals with sporadic Parkinson’s disease patients. This poses a problem because culturing patient-derived cell lines in parallel for such large cohorts requires specialist robotics, it is invasive for patients and is very expensive. To overcome this, we are collecting blood cells from a large cohort of Parkinson’s disease patients including sporadic and familial cases where mitochondrial proteins are affected. We will look for changes in mitochondria in these cells to try to identify subgroups of patients that may in future benefit from medicines that target mitochondrial dysfunction.
Schwarz, L., Casadei, N. and Fitzgerald, JC. (2021) Generation of R272Q, S156A and K572R RHOT1/Miro1 point mutations in iPSCs from a healthy individual using FACS-assisted CRISPR/Cas9 genome editing. Stem Cell Res. 25(55), 102469.
Brown SJ, Boussaad I, Jazaro J, Fitzgerald JC, Antony P, Keatinge M, Blechman, J, Schwamborn J, Krüger R, Placzek M, Bandmann O. PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons. Scientific Reports. SREP-20-02833A. (OA)
Körner, A., Bernard, A., Fitzgerald, J. C., Alarcon-Barrerer, J. C., Kostidis, S., Kaussen, T., Giera, M. & Mirakaj, V. 2021. Sema7A is crucial for resolution of severe inflammation. Proceedings of the National Academy of Sciences, 118, e2017527118.
Bus, C, Zizmare L, Feldkaemper M, Geisler S, Zarani M, Schaedler A, Klose F, Admard, J, Mageean CJ, Arena J, Fallier-Becker P, Ugun-Klusek A, Maruszczak K,Kapolou K, Schmid B, Rapaport D, Ueffing M, Casadei N, Krüger R, Gasser T, Vogt-Weisenhorn D, Kahle PJ, Trautwein C, Gloeckner CJ, Fitzgerald JC. (2020). Human Dopaminergic Neurons Lacking PINK1 Exhibit Disrupted Dopamine Metabolism Related to Vitamin B6 Co-Factors. iScience. Volume 23, ISSUE 12, 101797.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702004/
Hertlein V, Flores-Romero H, Das KK, Fischer S, Heunemann M, Calleja-Felipe M, Knafo S, Hipp K, Harter K, Fitzgerald JC, García-Sáez AJ. MERLIN: a novel BRET-based proximity biosensor for studying mitochondria-ER contact sites. Life Sci Alliance. 2019 Dec 9;3(1). pii: e201900600. doi: 10.26508/lsa.201900600. Print 2020 Jan.PMID:31818884 (OA)
Bus C, Geisler S, Feldkaemper M, Flores-Romero H, Schaedler A, Zittlau K, Zarani M, Uysal B, Casadei N, Fallier-Becker P, Schwarz L, Brouwers JF, Koch H, Ugun-Klusek A, Maruszczak K, Vogt-Weisenhor DM, Wurst W, Schmidt B Martens G, Brügger B, Rapaport D, Krüger R, Garcia A, Macek B, Gasser T, Kahle P, Fitzgerald JC. (2019). PINK1 Regulates Dopamine and Lipids at Mitochondria to Maintain Synapses and Neuronal Function bioRxiv MS: 814343.
Grossmann D, Berenguer-Escuder C, Bellet M, Scheibner D, Bohler J, Massart F, Rapaport D, Skupin A, Fouquier d'Hérouël A, Sharma M, Ghelfi J, Raković A, Lichtner P, Antony P, Glaab E, May P, Dimmer K, Fitzgerald JC, Grünewald A, Krüger R. (2019) Mutations in RHOT1 Disrupt Endoplasmic Reticulum-Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease. Antioxid Redox Signal. Dec 1;31(16):1213-1234. doi: 10.1089/ars.2018.7718. https://www.ncbi.nlm.nih.gov/pubmed/?term=31303019
Ugun-Klusek A, Theodosi TS, Fitzgerald JC, Burté F, Ufer C, Boocock D, Yu-Wai-Man P, Bedford L, Billett EE (Published online 2018, Oct 8). Monoamine oxidase-A promotes protective autophagy in human SH-SY5Y neuroblastoma cells through Bcl-2 phosphorylation. Redox Biol. 2019 Jan; 20: 167–181. https://www.ncbi.nlm.nih.gov/pubmed/30336354
Rotermund C, Machetanz G and Fitzgerald JC. (2018). The Therapeutic Potential of Metformin in Neurodegenerative Diseases. Front. Endocrinol. doi: 10.3389. Jul 19;9:400. https://www.ncbi.nlm.nih.gov/pubmed/30072954
Jores T, Lawatscheck J, Beke V, Franz-Wachtel M, Yunoki K, Fitzgerald JC, Macek B, Endo T, Kalbacher H, Buchner J, and Rapaport D. (2018). Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins. J. Cell. Biol. jcb.201712029; DOI: 10.1083/jcb.201712029. https://www.ncbi.nlm.nih.gov/pubmed/29930205
Fitzgerald JC, Zimprich A, Bobbili DR, May P, Sharma M, Krüger R. (2018). Reply: No evidence for rare TRAP1 mutations influencing the risk of idiopathic Parkinson’s disease. Brain. 141 (3):17 doi.org/10.1093/brain/awx380.
Sofi S, Fitzgerald JC, Jähn D, Dumoulin B, Stehling S, Kuhn H, Ufer C. (2018) Functional characterization of naturally occurring genetic variations of the human Guanine-rich RNA sequence binding factor 1 (GRSF1). Biochim. Biophys. Acta. 1862(4):866-876.
GMarrone L, Bus C, Schöndorf D, Fitzgerald JC, Kübler M, et al. (2018) Generation of iPSCs carrying a common LRRK2 risk allele for in vitro modeling of idiopathic Parkinson's disease. PLOS ONE. 13(3): e0192497.
Fitzgerald JC, Zimprich A, Carvajal Berrio DA, Schindler KM, Maurer B, Schulte C, Bus C, Hauser AK, Kübler M, Lewin R, Bobbili DR, Schwarz LM, Vartholomaiou E, Brockmann K, Wüst R, Madlung J, Nordheim A, Riess A, Martins LM, Glaab E, May P, Schenke-Layland K, Picard D, Sharma M, Gasser T, Krüger R. (2017). Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson’s disease. Brain. 140; 2444–2459.
Casadei N, Sood P, Ulrich T, Fallier-Becker P, Kieper N, Helling S, May C, Glaab E, Chen J, Nuber S, Marcus K, Rapaport D, Ott T, Riess O, Krüger R, Fitzgerald JC. (2016). Mitochondrial defects and neurodegeneration in mice overexpressing wild type or G399S mutant HtrA2. Human Molecular Genetics. 25(3):459-71. https://www.ncbi.nlm.nih.gov/pubmed/28031291
Burbulla LF, Fitzgerald JC, Stegen K, Westermeier J, Thost AK, Kato H, Mokranjac D, Sauerwald J, Martins LM, Woitalla D, Rapaport D, Riess O, Proikas-Cezanne T, Rasse TM, Krüger R. (2014). Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1. Cell Death Dis. 17;5:e1180.
Lisa Schwarz, University of Tübingen (2018-), Katharina Stegen, University of Tübingen (2013-2018), Christine Bus, University of Tübingen (with Thomas Gasser, 2013-2018).
Lara-Sophie Rieder, University of Tübingen (2020), Orchid Ammar, University of Tübingen (2020), Kim Krieg, University of Constance (2018-2019), Max Mattheuer, MCBI, University of Tübingen (2018), Anna Schaedler, GTC, Tübingen (2018), Konstantina Kopolou, GTC, Tübingen (2017), Dilara Halim, GTC, Tübingen (2017), Benedikt Hoelbling, GTC, Tübingen (2017), Marco Siekmann, GTC, Tübingen (2017), Lisa Schwarz, GTC, Tübingen (2017).
Kevin Schindler, University of Tübingen (2015), Rahel Lewin, University of Tübingen (2014)