Tideglusib Rescues Neurite Pathology of SPG11 iPSC Derived Cortical Neurons

Mutations in SPG11 cause a complicated autosomal recessive form of hereditary spastic paraplegia (HSP). Mechanistically, there are indications for the dysregulation of the GSK3β/βCat signaling pathway in SPG11. In this study, we tested the therapeutic potential of the GSK3β inhibitor, tideglusib, to rescue neurodegeneration associated characteristics in an induced pluripotent stem cells (iPSCs) derived neuronal model from SPG11 patients and matched healthy controls as well as a CRISPR-Cas9 mediated SPG11 knock-out line and respective control. SPG11-iPSC derived cortical neurons, as well as the genome edited neurons exhibited shorter and less complex neurites than controls. Administration of tideglusib to these lines led to the rescue of neuritic impairments. Moreover, the treatment restored increased cell death and ameliorated the membranous inclusions in iPSC derived SPG11 neurons. Our results provide a first evidence for the rescue of neurite pathology in SPG11-HSP by tideglusib. The current lack of disease-modifying treatments for SPG11 and related types of complicated HSP renders tideglusib a candidate compound for future clinical application.


INTRODUCTION
Hereditary spastic paraplegias (HSPs) are a heterogeneous group of motor neuron disorders. Clinically, HSP manifests with progressive lower limb spasticity and weakness due to axonopathy of corticospinal motor neurons and ascending dorsal columns. Up to date, more than 70 distinct genetic loci (Spastic Paraplegia Gene SPG1-SPG79) and mutations in more than 50 genes have been identified in HSP patients (Lo Giudice et al., 2014;Novarino et al., 2014). HSPs can be inherited in an autosomal dominant, autosomal recessive (AR), and, rarely, in an X-linked manner. Mutations in SPG11 are the most common genetic cause of AR complicated HSP. Apart from spastic paraparesis, SPG11 patients present with additional phenotypes that in the majority of cases include cognitive impairment, thin corpus callosum (TCC), neuropathy, and amyotrophy (Lo Giudice et al., 2014). Interestingly, mutations in SPG11 encoding spatacsin, were also found in other motor neuron diseases such as AR juvenile-onset amyotrophic lateral sclerosis (ALS5) and AR Charcot-Marie-Tooth disease (Montecchiani et al., 2016;Orlacchio et al., 2016). This indicates an important function of SPG11 in a variety of neuronal subtypes and shows that spatacsin causes multi system neurodegeneration.
Until now, more than 100 mutations in the 40-exon long SPG11 have been described. The majority of mutations result in premature stop codons, causing nonsense mediated RNA decay and/or truncated proteins. This implies a loss of function mechanism (Stevanin et al., 2013). SPG11 encodes spatacsin (a 2443 aa protein; ∼280 kDa), a potential transmembrane protein (Paisan-Ruiz et al., 2008) that has been implicated in axonal maintenance (Pérez-Brangulí et al., 2014). A number of studies have shown that the protein is linked to the autophagic-lysosomal machinery Renvoise et al., 2014;Varga et al., 2015;Branchu et al., 2017). In addition, siRNA mediated knock-down of SPG11, led to reduced neurite complexity of mouse dissociated cortical neurons (Pérez-Brangulí et al., 2014). Moreover, membrane-bound structures were observed within the processes of SPG11 patient, induced pluripotent stem cells (iPSC) derived cortical neurons (Pérez-Brangulí et al., 2014). This observation has been related to alterations in the transport of organelles, specifically anterograde transport and a lack of synaptic vesicle movement in SPG11 neurons (Pérez-Brangulí et al., 2014). Additionally iPSC-derived neural progenitor cells (NPCs) described a prominent neurodevelopmental defect. Reduced NPC proliferation was mediated by increased GSK3β activity followed by impairment of β-catenin signaling pathway (Mishra et al., 2016). Interestingly, administration of the specific GSK3β inhibitor tideglusib rescued the observed NPC proliferation defect (Mishra et al., 2016).
In the present study, we asked, whether tideglusib might not only improve proliferation, but might also have a positive effect on neurite pathology. We evaluated the effect of tideglusib on SPG11 patients' neurons. After confirming the presence of neurite pathology in iPSC derived cortical neurons from SPG11 patients and genome edited lines, we here established a treatment regimen for differentiated cortical neurons. By analyzing neuronal morphology and survival, we demonstrate that tideglusib is capable to reduce these neuronal impairments.

Patients
The patients included in this study (n = 3; Table 1) were female Caucasians with genetically confirmed compound heterozygous mutations in SPG11 (Hehr et al., 2007;Bauer et al., 2009;Pérez-Brangulí et al., 2014). SPG11-1 and SPG11-2 are sisters with a heterozygous nonsense mutation at c.3036C>A/p.Tyr1012X in exon 16 and a c.5798delC/p.Ala1933ValfsX18 mutation in exon 30. SPG11-3 has a heterozygous nonsense mutation at c.267G>A/p.Trp89X in exon 2 and a splice site mutation 1457-2A>G in intron 6. All patients were severely affected, also indicated by high scores on the Spastic Paraplegia Rate Scale (33-39 out of max. of 52; Schüle et al., 2006). All patients have a TCC accompanied by cognitive impairment, white matter lesions, cortical atrophy, as well as muscle wasting and motor-sensory neuropathy (Hehr et al., 2007). The controls (CTRL1; CTRL2) were age matched healthy Caucasian females with no previous history of movement or neurological disorders.

iPSC Derivation
Fibroblasts from SPG11 patients and controls were reprogrammed with retroviral transduction using the four Yamanaka factors (Klf4, c-Myc, Oct4, and Sox2) as previously described (Pérez-Brangulí et al., 2014). The Institutional Review Board approval (Nr. 4120: Generierung von humanen neuronalen Modellen bei neurodegenerativen Erkrankungen) and informed and written consent forms are on file at the Movement Disorder Clinic of the Department of Molecular Neurology, Universitätsklinikum Erlangen (Erlangen, Germany). All iPSC lines were screened for pluripotency and had a stable karyotype using the G-banding chromosomal analysis (data not shown). The SPG11 mutations were confirmed in the patient-derived lines. Two iPSC lines were used for each patient and control.

Targeted SPG11 Knock-Out With the CRISPR/Cas9 System
To generate the SPG11 knock-out line (cSPG11) we targeted exon 1 of SPG11 ( Figure 4A). The single gRNAs (sgRNA) (Sigma-Aldrich) were chosen with the CRISPOR web-tool 1 and cloned into the pX330 plasmid expressing SpCas9 (Addgene plasmid #42230) and the sgRNA according to the guidelines of the Zhang lab 2 . The cutting efficiency of the sgRNAs was evaluated in transfected 293T cells, by T7 endonuclease-mediated detection of insertions/deletions (indels) as previously described (Turan et al., 2016). The highly efficient sgRNA with an offtarget score of 92 (sequence at Supplementary Figure S3) was chosen for nucleofection of a human embryonic stem cell line (hESC; HUES6). All the experiments with HUES6 were conducted according to the German Stem Cell Act (RKI, 63. Genehmigung to BW). Forty-eight hours after nucleofection, cells were single cell sorted by flow cytometry, expanded and validated for homozygous knock-out by T7-endonuclease assay (Supplementary Figure S2A), sequencing (LGC genomics) and TIDE genotyping analysis 3 . The positive clones and isogenic control (clone that underwent the genome editing process but had no mutations) were evaluated for exonic off-target mutagenesis by PCR-mediated sequencing of potential offtarget sites of 5 candidate genes (selected with CRISPOR) (Supplementary Figure S3A). The genome edited lines were cultured on Matrigel (BD Biosciences) in mTeSR1 medium (Stem Cell Technologies). Detailed experimental scheme appears in Figure 4B.
as described previously ; Figure 1A). Briefly, pluripotent stem cells were cultured with mTeSR1 medium (Stemcell Technologies) and passaged using Gentle Cell Dissociation Reagent (Stemcell Technologies). For the generation of free floating embryoid bodies (EBs), they were incubated for 45 min at 37 • C with Collagenase IV and transferred to ultralow attachment plates (Corning) for 1 week. Afterwards, the EBs were transferred to polyornithine-laminin coated plates, followed by manual collection of neural rosettes. Rosettes were dissociated using TrypLE TM Express and transferred to proliferation media containing FGF 2 . For the induction of neuronal differentiation, the cells were cultured in N2/B27 media supplemented with the neurotrophins BDNF and GDNF, cAMP, and ascorbic acid. Tideglusib (Selleckchem; diluted in DMSO) was administered at the concentration of 1 µM to 1 week differentiated neurons and was applied twice a week, during media changes, for a period of 3 weeks (for details see paradigm Supplementary Figure S1).

Immunofluorescence and Image Analysis
For stainings, 100,000 NPCs were plated on 24-well plates with polyornithine-laminin coated glass cover slips and treated according to the paradigm in Figure 1A. To quantify neuronal cell death, PFA-fixed cells were pretreated with acetic acid and ethanol at a 1:2 ratio at −20 • C for 5 min. Afterward, the cells were stained with anti βIII-tubulin (Tuj-1; 1:500; BioLegend) and anti-cleaved-caspase3 (cCasp3; 1:500; Cell Signaling Technology) antibodies and mounted on microscope slides. Triplicate coverslips were used for each cell line and three random images per coverslip with comparable cell densities (40×) were acquired using Observer.Z1 fluorescence microscope (Zeiss). Representative images were obtained with LSM-780 confocal microscope setups (Carl Zeiss). For additional stainings, anti-glial fibrillary acidic protein (1:500; DAKO), and anti-Ctip2 (1:300; Abcam) antibodies were used. The quantifications were conducted with the cell counter plugin of Fiji software (Schindelin et al., 2012).

Gene Expression Analysis
The RNA was extracted using RNeasy kit (Qiagen) according to the manufacturer's instructions. A total of 500 ng RNA was reverse-transcribed into cDNA in 20 µl reaction solution by QuantiTect Reverse Transcription Kit (Qiagen). Subsequently, 1 µl of cDNA was used for real-time polymerase chain reaction (qPCR). The qPCR program definitions were as follows: 95 • C for 10 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min and 1 cycle at 95 • C for 15 s, 60 • C for 30 s and 95 • C for 15 s. The primers are listed in Supplementary Table S1.

Neurite Length and Complexity Analysis
Neurons derived from pluripotent stem cells were cultured in 24-well plates, on top of polyornithine-laminin coated glass coverslips. In order to allow visualization of single neurons, on day 26 the cells were transfected with pEF1-dTomato using Lipofectamine, 2000 (Invitrogen, reagent:DNA ratio 2:1). The cells were fixed 48 h after transfection with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The cells were stained for neuronal marker expression using the anti-βIII-tubulin antibody on coverslips (Tuj-1; 1:500; BioLegend), and the coverslips were mounted on slides. Images were captured with a 10× objective lens using the TILE function of the Zen Pro Software (Zeiss) on the Observer.Z1 fluorescence microscope (Zeiss). Semi-automated tracing of individual transfected βIIItubulin positive cells was performed using the NeuronJ plugin of Fiji (Schindelin et al., 2012). A minimum of 36 cells per patient/control and 18 cells for genome edited lines were analyzed for the number of neurites and total neurite length. Furthermore, semi-automated Sholl analysis was carried out at predefined 50 µm intervals from the soma using the Fiji software.

Electron Microscopy
Transmission electron microscopy was performed as described previously (Schlotzer-Schrehardt et al., 2012;Havlicek et al., 2014). Briefly, the neurons were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Subsequently, cells were post-fixed in 2% buffered osmium tetroxide and dehydrated in graded alcohol concentrations. Afterward, the cells were embedded in epoxy resin. Horizontal sections were stained with uranyl acetate and lead citrate. Finally, the sections were examined using a transmission electron microscope (LEO 906E; Carl Zeiss Microscopy).

Statistical Analysis
All statistical analyses were performed using IBM SPSS statistics software (version 23). The Student two tailed t-test for unpaired variables was applied when comparing the means between two groups. For comparison of more than three groups, one-way ANOVA followed by Bonferroni post hoc test was applied. P-values ≤ 0.05 were considered statistically significant. Unless indicated otherwise, all data are shown as mean ± SEM.

Characterization of iPSC Derived Neurons
The neuronal differentiation (described in Figure 1A) yields in cortical neurons expressing a majority of Tuj-1 positive cells with astrocytes comprising less than 10% of the cellular population ( Figures 1B,C). There was no significant difference in the amount of astrocytes between SPG11 lines and controls ( Figure 1C). Immunofluorescence stainings as well as qPCR analysis confirm the neuronal cortical identity of the iPSC derived cells (Figures 1D,E). Thus, both controls and SPG11 cell lines exhibited a significantly higher transcript level of the deep layer marker Ctip2 and the vesicular glutamate transporter 1 (VGLUT1) compared to iPSC lines ( Figure 1D). The gene expression levels did not differ between patients and controls ( Figure 1D).

Rescue of SPG11 Neurite Outgrowth Abnormalities by Tideglusib Treatment
We first evaluated the neurite morphology of SPG11 iPSC derived neuronal cultures and measured neuritic length and the number of neurites per cell, indicative of neuritic complexity (Figure 2A). Compared to CTRLs, neurite length was significantly decreased by 78% in SPG11 (669.44 ± 50.34 µm in SPG11, compared to 3103.48 ± 328.04 µm in CTRL; P ≤ 0.05; Figure 2B). Moreover, neurite number was significantly reduced (2.87 ± 0.16 in SPG11, compared to 6.76 ± 1.0 in CTRL; P ≤ 0.05; Figure 2C). These results are in agreement with the previously described neuritic impairment in SPG11 iPSC derived neuronal cultures (Pérez-Brangulí et al., 2014). We next tested the effect of the chronic administration of 1 µM tideglusib. The rationale for this dose was obtained from previous experiments, which revealed severe toxicity at a higher dose (5 µM; data not shown). We applied the compound twice a week for a period of 3 weeks until neurons reach maturity (see paradigm Supplementary Figure S1). Interestingly, chronic administration of tideglusib was able to significantly rescue neurite length (2856.86 ± 151.65 µm in SPG11 treated with tideglusib, compared to 3103.48 ± 328.04 µm in CTRL; P ≤ 0.05; Figure 2B). Moreover, neurite number was restored to a level which was virtually comparable to CTRL (6.48 ± 0.64 in SPG11 treated with tideglusib, compared to 6.76 ± 1.04 in CTRL; P ≤ 0.05; Figure 2C).
In order to further elaborate the effect of the chronic administration of tideglusib on neurite complexity, Sholl analysis was performed. The number of intersections of neurites at various radial distances from the cell soma was measured and revealed a significant increase in neuritic arborization of SPG11 patient derived neurons upon tideglusib treatment ( Figure 2D). This effect was significant for all distances' measures (distances from soma from 10 µm to 1660 µm, P ≤ 0.05).
The ultrastructural analysis of SPG11 iPSC derived 4-weeks differentiated neurons revealed that the neurites accumulate membrane-bound inclusion bodies measuring 0.3-1.5 µm in diameter with electron-dense contents. Interestingly, tideglusib treatment reverted this phenotype and led to an elimination of these abnormal structures ( Figure 3E).

CRISPR/Cas9 Mediated Generation and Characterization of SPG11 Knock-Out Line
Sequencing analysis of HUES6 line transfected with a plasmid containing Cas9 and gRNA targeting exon 1 of SPG11 revealed one clone with out-of-frame mutation (cSPG11, Figures 4C,D). According to TIDE analysis, the clone was positive for out-offrame mutation in both alleles (Figures 4C,D). An additional clone, that underwent the genome editing process, but had no mutations (cCTRL; Figure 4D), was chosen as isogenic control. Both cSPG11 and cCTRL were negative for off-target activity (Supplementary Figures S3A,B). The reduction of spatacsin in cSPG11 confirms the efficiency of the genome editing strategy (Supplementary Figure S2B). The residual amount of the protein can be attributed to the lack of specific and reliable antibodies for spatacsin detection. Genome edited pluripotent stem cells were differentiated according to the paradigm in Figure 1A and were positive for Tuj1 ( Figure 4E) with astrocytes levels that, similarly to iPSC-derived neuronal cultures, were lower than 10% and did not differ between cSPG11 and cCTRL (Supplementary Figure S2C).

DISCUSSION
Our findings indicate a beneficial effect of tideglusib on both cellular morphology and survival of SPG11-iPSCs-derived neurons. This is the first indication of an effective therapeutic approach applied to human SPG11 differentiated neurons.
First, our data confirm the findings from a previous study (Pérez-Brangulí et al., 2014) that SPG11 neurons present with impaired neuritic length and decreased number of neurites, indicative of reduced neuronal complexity. Our study was able to rescue these impairments by administration of the irreversible GSK3 inhibitor, tideglusib, to neuronal cells. In addition, we were able to recapitulate virtually similar neuritic impairments, followed by tideglusib rescue in CRISPR/Cas9 mediated SPG11 knock-out line. Previous findings revealed that SPG11 NPCs exhibit dysregulation of GSK3β/βCat signaling pathway (Mishra et al., 2016). The data of our current study indicate that this pathway has a prominent role not only during neural development, but also in mature neurons. We are able to show, that in SPG11 tideglusib has a significant role in rescuing the neuritic morphology.
Increased activity of GSK3β has been implicated in neurodegeneration, particularly in Alzheimer's Disease (AD). Animal models overexpressing GSK3β exhibit one of the main AD hallmarks -increased tau phosphorylation (Brownlees et al., 1997;Lucas et al., 2001), whereas overexpression of GSK3β in the dentate gyrus leads to neurodegeneration of this area (de Barreda et al., 2010). Tau hyper-phosphorylation, elevated amyloid levels and activation of GSK3β are also present in AD-iPSC derived neurons (Ochalek et al., 2017). Conversely, inhibition of GSK3β leads to reduction of β-amyloid production (Rockenstein et al., 2007) and its toxicity (Koh et al., 2008), reduction of phosphorylated tau in cultured neurons (Zhang et al., 2011) and improves learning and memory in AD mouse models (Farr et al., 2016). Although the onset and patterns of cognitive symptoms and cortical atrophy present in SPG11 patients are markedly different from those observed in AD patients, our data suggest that GSK3β overactivation may be a common molecular denominator of both diseases.
We next found that tideglusib treatment was able to rescue the increased cell death of SPG11 neurons. The potential of tideglusib to diminish apoptosis in SPG11 was first revealed in a study where tideglusib administration to SPG11-NPCs improved their survival and proliferation (Mishra et al., 2016). Here, we extended the therapeutic value of tideglusib to neurons by showing for the first time, that differentiating neurons are also responsive to its effects. Our results are compatible with the data indicating the important role of GSK3β in the regulation of apoptosis (Jacobs et al., 2012). In addition, it has been shown that GSK3β inhibition leads to neuroprotective effects (Culbert et al., 2001).
Moreover, in rat cerebellar granule neurons cultures GSK3β inhibition lead to increase of neuronal survival (Song et al., 2010), whereas in a mouse model of CNS injury it promoted axonal regeneration (Song et al., 2010;Guo et al., 2016). Overall, these studies conclude that GSK3β inhibition has a beneficial effect on mature neurons which also provides an explanation to the observed rescue of SPG11 neurodegenerative phenotypes by tideglusib.
Interestingly, SPG11 knock-out line showed no difference in cell death, and the number of cCas3 positive cells was not changed following tideglusib treatment. This result may arise from the vast heterogeneity and lack of correlation between genotype and phenotype in SPG11. Thus, in addition to HSP type 11, mutations in SPG11 can lead to autosomal recessive juvenile amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease (Montecchiani et al., 2016). This observation indicates the importance of the use of patient specific lines for disorders with complex genotypes.
An additional important observation is that tideglusib treatment leads to reduction of membranous inclusion bodies in SPG11 neurites. The possibility to reduce this impairment emphasizes the potency of tideglusib as a putative therapeutic compound.
Overall, our results increase the clinical relevance of the compound tideglusib and its potential to serve as a therapeutic agent for SPG11. Furthermore, they contribute to the establishment of iPSCs as a potent tool for disease modeling and drug testing. The fact that tideglusib has been approved by FDA and has been tested in clinical trials of Alzheimer's Disease, Autism Spectrum Disorder and Myotonic Dystrophy renders a potential availability also for SPG11 patients. Future studies that will utilize the advanced cerebral organoid models and delve into the mechanistic changes occurring after compound administration are essential for providing a robust basis for clinical implementation.

ACKNOWLEDGMENTS
This work is dedicated to our patients and, in particular, to the memory of Anne Wahlig. The present work was performed in fulfillment of the requirements for obtaining the degree "Dr. med." (AS). We would like to thank Himanshu Mishra and Francesc Perez-Branguli for their valuable contributions and to Holger Wend for excellent technical support.