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Rapid semi-automated phenotypic assays for compound testing in patient-derived SPG4 neurons

dc.contributor.advisorBrüstle, Oliver
dc.contributor.authorRehbach, Kristina
dc.description.abstractHereditary spastic paraplegia (HSP) is an inherited disease characterized by progressive spasticity in the lower limbs, caused by axonal degeneration of corticospinal motor neurons. Spastic paraplegia 4 (SPG4) is the most frequent, autosomal dominant subtype, responsible for >50% of all pure HSP cases. Affected patients carry mutations in the SPAST gene encoding the microtubule-severing enzyme spastin. So far, no curative treatment for HSP is available and drug discovery screens are hampered by the lack of suitable model systems. While SPG4-associated phenotypic alterations have been described in iPSC-derived neurons, development of these in vitro phenotypes typically requires several weeks of in vitro differentiation, limiting their exploitation for high-throughput assays. Therefore, developing a SPG4 model and enabling rapid phenotypic analyses within a few days is of great interest and became the focus of this study. To this end, fibroblasts of family members carrying a specific SPAST nonsense mutation were reprogrammed to a pluripotent state employing retroviruses or non-integrating Sendai viruses encoding OCT4, KLF4, SOX2 and c-MYC yielding in several fully validated SPG4 iPSC lines. IPSCs from three patients carrying heterozygous SPAST nonsense mutations were differentiated into highly enriched neuronal cortical cultures comprising >80% glutamatergic neurons expressing the layer V/VI markers CTIP2 and TBR1. Spastin levels in SPG4 neuronal cultures were reduced by approximately 50% compared to controls. Focusing on the identification of early neuronal HSP-related phenotypes, SPG4 neurons exhibited a 51% reduction in neurite length compared to controls already 24 hours after plating. At that time point, enlarged growth cones suggestive of a cytoskeletal imbalance were observed as well. Moreover, axonal swellings a hallmark of the HSP pathology, could be reliably detected already five days after plating of SPG4 iPSC-derived cortical neurons. Swellings were 1-7µm in diameter and stained positive for the axonal markers TAU1 and acetylated tubulin. Furthermore, these disease specific early phenotypes appeared to be cell type specific and could not be found in GABAergic SPG4 forebrain neurons, which might be due to a higher expression of M1 SPAST in this cell type. However, the application of different read-through inducing molecules did not lead to an up-regulation of spastin levels in patient cultures. To identify new potentially therapeutic compounds, counteracting SPG4-associated neuronal phenotypes, all three fast phenotypic assays were transferred to an automated or semi-automated 96-well-setup. Indeed, the actin-destabilizing drug Latrunculin B and the liver X receptor (LXR) agonist GW3965 led to a significant increase in patient neurite length. And eight of the tested drugs achieved a significant reduction of patient growth cone areas, including Latrunculin B and GW3965. The most effective reduction of axonal swellings, accompanied by normal neuronal morphology was achieved by the bone morphogenetic protein (BMP) inhibitor DMH1 and the LXR agonist GW3965. In particular, GW3965 was able to rescue all three phenotypes of SPG4 neurons and had no effect on control neurons. In summary, in this thesis several rapid phenotypic assays for disease modeling and drug screening in SPG4 neurons have been developed. In addition, the cortical neurons generated in this thesis are cryopreservable and prepared cell batches are readily available for future screening purposes. Taken together, the findings of this thesis provide an excellent basis for studying the underlying pathomechanisms as well as for drug development in hereditary spastic paraplegia.
dc.rightsIn Copyright
dc.subjectDisease modeling
dc.subjectstem cells
dc.subject.ddc570 Biowissenschaften, Biologie
dc.titleRapid semi-automated phenotypic assays for compound testing in patient-derived SPG4 neurons
dc.typeDissertation oder Habilitation
dc.publisher.nameUniversitäts- und Landesbibliothek Bonn
ulbbnediss.affiliation.nameRheinische Friedrich-Wilhelms-Universität Bonn
ulbbnediss.instituteMedizinische Fakultät / Institute : Institut für Rekonstruktive Neurobiologie (IRN)
ulbbnediss.fakultaetMathematisch-Naturwissenschaftliche Fakultät
dc.contributor.coRefereeWitke, Walter

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