The hypothesis of this project is that the different types of autosomal recessive complicated hereditary spastic paraplegia (HSP) share the same pathophysiology of changes in the GSK3 signalling pathway. If confirmed, this pathway might allow a common therapeutic approach these genetically distinct subtypes of HSP. Using human neuronal models for SPG11 and SPG15 differentiated from pluripotent stem cells after gene knockout by CRISPR/Cas9, we will investigate to what extent the GSK3 pathway contributes to neurodegeneration in different autosomal recessive HSP types. We will also investigate changes in the GSK3 pathway in other neuronal cell types relevant for the disease (alpha motoneurons, sensory neurons). In addition, we will test whether GSK3 inhibition can normalize neurodegeneration in CRISPR/Cas9-modified neurons and in patient-specific neurons.

These experiments will allow us to specify neuronal cell types that are dependent on the GSK3 signaling pathway. In addition, mechanisms of action of GSK3 inhibition in autosomal recessive HSP will be identified. Finally, the project aims at predicting therapy responses in complicated HSP types.

In subproject 7, human patient-derived cellular models of autosomal recessive complicated HSP were established and investigated with regard to molecular changes and potential therapeutic targets with a focus on the GSK3 signaling pathway. The following findings were obtained:

(1) Investigation of further neural cell types

Autosomal recessive complicated HSPs are complicated HSP types, i.e. multiple neural systems are affected by progressive neurodegeneration. In previous studies, only one disease model was used in forebrain models (including forebrain progenitor cells and cortical forebrain neurons). In the present project, the differentiation of patient-specific induced pluripotent stem cells (iPSCs) into other neural cell types was successfully established: First, spinal (alpha) motor neurons, which are also affected in autosomal recessive complicated HSP, were generated with high efficiency. This revealed similar degenerative features as in cortical neurons, with mitochondrial-axonal abnormalities in particular being identified (Güner et al., Front Neurosci 2021). This disease model of the lower motor neuron will therefore be available for future pharmacological studies in addition to the forebrain models (Pozner et al., Brain 2020). Glial cells are also significantly involved in neurodegenerative diseases, but have so far been insufficiently considered in stem cell models. In addition, the HSP-associated genes including SPG11 and SPG15 are also expressed in glial cells. To incorporate this cellular heterogeneity of the CNS and to shed light on the role of cell-cell interactions (Simmnacher et al., Front Cell Neurosci 2020; Krach et al., Mol Cell Neurosci 2020), a differentiation protocol into induced microglia-like cells was successfully established, which forms the starting point for the investigation of immune-mediated mechanisms in HSP subtypes (Lanfer et al., Int J Mol Sci 2022; Regensburger et al., Brain Behav Immun 2023).

(2) GSK3 signaling pathway validation

Based on previous data of GSK3 overactivation in SPG11 forebrain models, the SPG15 genotype was investigated for the first time in the present project. Patient-specific iPSCs from HSP patients with the SPG15 genotype were successfully reprogrammed into iPSCs, which was confirmed by pluripotency analyses. For a valid comparison to healthy control iPSCs and SPG11 iPSCs, all three groups were differentiated into neural progenitor cells and finally mature neurons. On the one hand, overactivation of the GSK3 pathway was also demonstrated as a therapeutic target in SPG15, which allowed the effects of treatment with a GSK3 inhibitor to be characterized. The effects were not only described for the SPG15 genotype, but also independently confirmed previous data for the SPG11 genotype.

(3) Transcriptional dysregulation

A third aim of the project was a transcriptional characterization. While classical transcriptome analyses have so far been restricted to comparing expression levels of individual genes, the bioinformatic orientation of the present project enabled the establishment of an analysis pipeline for differential mRNA splicing. Its successful application in the iPSC model led to the identification of a new splicing modulator in motor neuron diseases (Krach et al., Acta Neuropathol 2022). In addition, an innovative approach showed that the present analysis pipeline can efficiently reveal effects of small molecules as a novel CNS-targeted therapy in the human model (Krach et al., Nat Commun 2022). This breakthrough opens up the possibility of RNA-based drug screening in other genotypes of HSP and in neurogenetic diseases in general.

(4) Role of other cellular mechanisms

The pathophysiology of autosomal recessive complicated forms of HSP has been linked to several signaling pathways, including GSK3, lysosomal-autophagic degradation pathways, and mitochondrial-endoplasmic reticulum (ER) interaction. The ER plays a central role in the interaction of the individual organelles and exhibits high-resolution signaling flexibility with regard to calcium metabolism. In the present project, “store operated calcium entry” was established in both non-neuronal and neuronal cell models and will also serve as a screening platform for active substances in the future (Rizo et al., Brain 2022). In the present project, further analyses of lysomal and neurodegeneration-associated proteins were advanced as a secondary finding, which formed the starting point for projects in the subsequent funding period (Regensburger et al., Mov Disord 2020; Regensburger et al., Front Cell Dev Biol 2020; Leupold et al., Front Neurol 2022).

(5) Gene-based labeling of HSP-associated proteins

A major obstacle to research into autosomal recessive HSP types has been the lack of specific antibodies for valid detection of involved proteins by Western blot or immunohistochemistry. With the aid of CRISPR/Cas9 technology, various cell models were therefore modified in the present project so that the SPATACSIN protein, which is encoded by the SPG11 gene, is provided with a C-terminal marker (Krumm et al., Stem Cell Res 2021). The use of these cell lines will facilitate the investigation of the localization and protein expression levels in the future. In addition, the method can also be applied to other HSP-associated proteins in the future.

(6) Translational patient-specific research aspects

A significant strength of the research institution is the close association of patient data with phenotyping results of the respective patient-specific cell models in the sense of personalized medicine (bedside-to-bench). This requires, on the one hand, an exact clinical follow-up of the cohort and, on the other hand, the development of new disease parameters. This is the only way to ensure that in vitro results can be transferred to patients in the future (bench-to-bedside). As a secondary result of this continuous monitoring of the clinical cohort, new clinical markers in imaging, neuropsychology and metabolic processes have been successfully identified (Regensburger et al., BMC Neurol 2020; Utz et al., Orphanet J Rare Dis 2022; Regensburger et al., Nutrients 2022).