GSK3ß-dependent dysregulation of neurodevelopment in SPG11-patient induced pluripotent stem cell model

Abstract

Objective: Mutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal-recessive complex hereditary spastic paraplegia (HSP) and juvenile-onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient-specific induced pluripotent stem cell (iPSC)-derived cortical neural progenitor cells (NPCs).

Methods: We generated and characterized iPSC-derived NPCs and neurons from 3 SPG11 patients and 2 age-matched controls.

Results: Gene expression profiling of SPG11-NPCs revealed widespread transcriptional alterations in neurodevelopmental pathways. These include changes in cell-cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß-signaling pathway was found to be dysregulated in SPG11-NPCs. Impaired proliferation of SPG11-NPCs resulted in a significant diminution in the number of neural cells. The decrease in mitotically active SPG11-NPCs was rescued by GSK3 modulation.

Interpretation: This iPSC-derived NPC model provides the first evidence for an early neurodevelopmental phenotype in SPG11, with GSK3ß as a potential novel target to reverse the disease phenotype. Ann Neurol 2016;79:826-840.

Figure 1

Generation of iPSCs from SPG11 patients and controls (CTRL). (A) MRI analysis of CTRL and SPG11 patients included in the study. (B) Pedigrees of SPG11 families. Female index patients are represented in black circles. (C) Mutations analysis in SPG11 patient fibroblasts. Patients 1 and 2 (SPG11‐1, SPG11‐2) have heterozygous nonsense mutations at c.3036C > A/p.Tyr1012X in exon 16 and c.5798 delC/p.Ala1933ValfsX18 in exon 30. Patient 3 (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. (D) Schematic representation of the neuronal differentiation paradigm. Scale bar = 50µm. iPSCs = induced pluripotent stem cells; MRI = magnetic resonance imaging; NPCs = cortical neural progenitor cells.

Figure 2

Global gene expression analysis of day 3 NPCs generated from SPG11‐ and CTRL‐iPSCs. n = 2 samples from each individual. (A) Heatmap showing hierarchical clustering of differentially expressed genes in SPG11‐NPCs compared to CTRL‐NPCs. (B) Histographs of total number of differentially expressed genes (upregulated genes in red, downregulated genes in green; p ≤ 0.05). (C) Gene Ontology term analysis of important biological processes enriched within differentially expressed genes (p values: Benjamini‐Hochberg corrected). (D) Wnt/GSK3ß pathway‐related genes differentially regulated in SPG11‐NPCs. (E) Cell‐cycle–related genes differentially regulated in SPG11‐NPCs. (F) Cortical development‐related genes differentially regulated in SPG11‐NPCs. (G) List of differentially regulated transcripts related to autophagy, endolysosomal, and ER stress pathways. Upregulated transcripts shown in red, downregulated transcripts shown in green (p ≤ 0.05), analyzed by one‐way ANOVA (D–G). (H) Validation of transcriptome data with qRT‐PCR in SPG11‐ and CTRL‐NPCs. Table showing fold‐change difference for 10 selected genes of Wnt/GSK3 signaling‐, cell‐cycle–, cortical development‐, and autophagy‐related pathways (p ≤ 0.05). (I) Bar graph showing relative gene expression of the 10 selected genes. Relative expression shows the average values of four CTRL‐NPCs (set at a value of 1) and six SPG11‐NPC lines. mRNA levels were normalized against GAPDH. Data are represented as mean ± SEM; *p ≤ 0.05 by two‐tailed Student t test. APC = adenomatous polyposis coli, TCF7L2 = transcription factor 7‐like 2, FOSL2 = Fos‐related antigen 2, CCNA1 = cyclin‐A1, CDH1 = cadherin‐1, ITSN1 = intersectin‐1, CDC42EP3 = Cdc42 effector protein 3, SEM3A = semaphorin‐3A, LYST = lysosomal trafficking regulator. ANOVA = analysis of variance; ER = endoplasmic reticulum; GAPDH = glyceraldehyde 3‐phosphate dehydrogenase; iPSCs = induced pluripotent stem cells; MRI = magnetic resonance imaging; mRNA = messenger RNA; NPCs = cortical neural progenitor cells; qRT‐PCR = quantitative real‐time polymerase chain reaction.

Figure 3

Reduced proliferation and neurogenesis in SPG11‐NPCs. (A) Representative images of Nestin/Sox2 double‐positive NPCs generated from SPG11- and CTRL-iPSCs. Nuclei were visualized with DAPI. Scale bar = 50µm. (B) SPG11‐NPCs exhibit a decreased Nestin/Sox2 cell density compared to CTRL-NPCs. (C) No difference in Nestin/Sox2 double‐positive cells (% over DAPI) between SPG11- and CTRL-NPC lines. (D) Differentiated neuronal cells expressing neuron‐specific (Tuj1) and glia‐specific (GFAP) markers. Nuclei were visualized with DAPI. Scale bar = 50µm. (E) SPG11‐NPCs exhibit a marked reduction in neuronal cell density compared to CTRL. (F) SPG11‐NPCs show reduced generation of Tuj1‐positive neurons compared to CTRL-NPCs, reflecting neurogenesis deficits in SPG11 patients. Data are represented as mean ± SEM. ^**p < 0.01; ^***p < 0.001, by one‐way ANOVA followed by Dunnett's post‐hoc multiple comparison test (B, C, E, F). ANOVA = analysis of variance; DAPI = 4’,6‐diamidino‐2‐phenylindole; GFAP = glial fibrillary acidic protein; iPSCs = induced pluripotent stem cells; NPCs = cortical neural progenitor cells.

Figure 4

SPG11‐NPCs show altered cell‐cycle distribution and stage‐specific downregulation of important checkpoint genes. (A) Representative images of Nestin/Sox2‐positive cells colabeled with BrdU‐positive nuclei for SPG11‐ and CTRL‐NPCs. Nuclei were visualized with DAPI. Scale bar = 50µm. (B) SPG11‐derived Nestin/Sox2‐positive cells have significantly reduced numbers of BrdU‐labeled cells compared to CTRLs. (C) SPG11‐ and CTRL‐NPCs (Nestin/Sox2⁺) colabeled with the endogenous proliferation marker, PCNA. Nuclei were visualized with DAPI. Scale bar = 50µm. (D) SPG11‐derived Nestin/Sox2 double‐positive NPCs have significantly reduced numbers of PCNA colabeled cells. (E) Diminished number of Nestin/Sox2‐positive NPCs colabeled with mitotic marker H3P of SPG11‐NPCs, suggesting a compromised mitosis. (F) Schematic representation of important checkpoint and senescence genes of the cell cycle. CDK = cyclin‐dependent kinase; p21^Cip1 = cyclin‐dependent kinase inhibitor 1; p27^Kip1 = cyclin‐dependent kinase inhibitor 1B. (G–K) Flow cytometry/PI staining analysis shows the respective distribution of cells in G₁, S, and G₂/M phases of the cell cycle for SPG11‐ and CTRL‐NPC lines. (I) No significant difference in the percentage of viable cells in the G₁ phase of cell cycle between SPG11‐ and CTRL‐NPCs. However, SPG11‐NPCs have a highly diminished percentage of viable cells in the S phase (J) and G₂/M phase (K). Data represented as mean ± SEM: ^**p < 0.01; ^***p < 0.001, by one‐way ANOVA followed by Dunnett's post‐hoc multiple comparison test (B, D, E, I–K). (L–O) mRNA expression analysis of SPG11- and CTRL-NPCs revealed no significant difference for G₁ phase cell‐cycle checkpoint markers CDK4 (L) and CDK6 (M). ns = not significant. (N) Expression of cell‐cycle genes at the S phase CDK2 is significantly downregulated in SPG11‐NPCs. (O) Significant downregulation of G₂/M phase cell‐cycle gene CDK1 highlighted a perturbed cell‐cycle activity in SPG11‐NPCs. mRNA levels were normalized against GAPDH. (P–R) Protein expression of CDK2 and CDK1 using Western blot analysis in SPG11‐NPCs compared to CTRL‐NPCs. Significant decrease in CDK2 protein levels (Q) and CDK1 (R) in SPG11‐NPCs compared to the CTRL. CDK2 and CDK1 expression was normalized against GAPDH. (S–T) Increased rate of apoptosis in SPG11‐NPCs. Immunofluorescent images of SPG11‐ and CTRL‐NPCs stained with apoptotic marker cleaved caspase 3. (S) SPG11‐NPCs have 2‐ to 3‐fold increase in number of apoptotic cells compared to CTRL-NPCs (T). Nuclei were visualized with DAPI. Scale bar = 20µm. Data represented as mean ± SEM: ^*p < 0.05; ^**p < 0.01, by two‐tailed Student t test (L–O, Q, R, T). ANOVA = analysis of variance; BrdU = 5‐bromo‐2’‐deoxyuridine; cleav. Casp3 = cleaved caspase‐3; DAPI = 4’,6‐diamidino‐2‐phenylindole; GAPDH = glyceraldehyde 3‐phosphate dehydrogenase; H3P = phospho‐histone 3; mRNA = messenger RNA; NPCs = cortical neural progenitor cells; PCNA = proliferating cell nuclear antigen.

Figure 5

Increased GSK3ß activity leads to reduced ß‐Catenin levels in SPG11‐NPCs. (A) Phospho‐GSK3ß (Ser9) protein expression in SPG11‐ and CTRL‐NPC lines. (B) Protein expression of p‐GSK3ß (Ser9) was significantly reduced in SPG11‐NPCs compared to CTRL‐NPCs. p‐GSK3ß expression was normalized against total GSK3ß. (C) ß‐Catenin protein levels in SPG11‐ and CTRL‐NPC lines. (D) Significant decrease in the ß‐Catenin protein levels in SPG11‐NPCs compared to the CTRL. ß‐Catenin expression was normalized against GAPDH. (E) Schematic representation of ß‐Catenin (TCF/LEF) reporter activity assayed using TOP/FOP flash luciferase assay. (F) TOP flash luciferase activity is 2‐fold reduced in SPG11‐NPCs compared to the CTRL‐NPCs under normal conditions. (G) Wnt pathway activation by treatment with Wnt3a rescues ß‐Catenin signaling mediated TCF/LEF reporter activity in SPG11‐NPCs; n = 3. Data represented as mean ± SEM: ^* p < 0.05, by two‐tailed Student t test (F, G). (H) Representative Western blot for expression of senescence marker p27^Kip1 in SPG11‐ and CTRL‐NPC lines. (I) Two‐ to three‐fold increase in p27^Kip1 protein levels in SPG11‐NPCs compared to CTRL‐NPCs. Data represented as mean ± SEM: n = 3; ^* p < 0.05; ^** p < 0.01, by one‐way ANOVA followed by Dunnett's post‐hoc multiple comparison test (B, D, I). ANOVA = analysis of variance; GAPDH = glyceraldehyde 3‐phosphate dehydrogenase; NPCs = cortical neural progenitor cells. See also Supplementary Table 1. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

Figure 6

GSK3 antagonists (CHIR99021 and tideglusib) rescue neurodevelopmental defects of SPG11‐NPCs. (A) NPCs were treated with 3µM of the GSK3 inhibitor (tideglusib) for 24 hours. Representative images of untreated SPG11‐NPCs (SPG11‐NT) and tideglusib‐treated SPG11‐NPCs (SPG11‐Tide) on day 3. Cell proliferation was analyzed using colabeling of PCNA in Nestin/Sox2‐positive NPCs. Nuclei were visualized with DAPI. Scale bar = 50µm. (B) Increased numbers of Nestin/Sox2‐positive cells colabeled with PCNA in CHIR99021‐treated SPG11‐NPCs. (C) Tideglusib‐treated SPG11‐NPCs, compared to untreated NPCs, revealed restoration of cell proliferation similar to the CTRL‐NPCs. (D) ß‐Catenin protein levels in tideglusib‐treated group (CTRL‐Tide/SPG11‐Tide) and untreated group (CTRL‐NT/SPG11‐NT). (E) Almost 2‐fold increased expression of ß‐Catenin protein levels in Tide‐treated SPG11‐NPCs (SPG11‐Tide) compared to the untreated group (SPG11‐NT). ß‐Catenin expression was normalized against γ-Adaptin. (F) TOP flash luciferase activity is almost 2‐fold enhanced in SPG11‐NPCs treated with tideglusib, suggesting restoration of Wnt/ß‐Catenin signaling in SPG11‐NPCs. (G, H) Representative mRNA expression profile of important cell‐cycle checkpoint genes, CDK2 and CDK1 in SPG11‐ and CTRL‐NPCs treated with tideglusib. (H) Almost 2‐fold increase in mRNA expression of S phase marker (CDK2) and more than 3‐fold increase in expression of G₂/M phase marker (CDK1) in SPG11‐NPCs treated with tideglusib (SPG11‐Tide). (I) Neural differentiation in presence of 3µM of the GSK3 inhibitor (tideglusib) increased the numbers of neural cells in tideglusib‐treated SPG11‐NPCs, compared to untreated NPCs. Data represented as mean ± SEM: ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p < 0.001, by two‐tailed Student t test; n = 3 (B, C, E–I). ß‐Catenin‐NT = ß‐Catenin expression in nontreated NPCs; ß‐Catenin‐Tide = ß‐Catenin expression in tideglusib‐treated NPCs, γ‐Adaptin‐NT = γ‐Adaptin expression in nontreated NPCs; γ‐Adaptin–Tide = γ‐Adaptin expression in tideglusib‐treated NPCs; ANOVA = analysis of variance; DAPI = 4’,6‐diamidino‐2‐phenylindole; mRNA = messenger RNA; NPCs = cortical neural progenitor cells; PCNA = proliferating cell nuclear antigen.

Figure 7

(A) Proposed two distinct stages of SPG11 pathogenesis. Neurodevelopmental phenotype represents early onset within the first two decades characterized by a proliferation deficit, impaired cortical development and consequently to cognitive impairment. The neurodegenerative phenotype, marked by progressive spasticity and paraparesis, leads to functional neuronal deficits, motor neuron degeneration, and peripheral sensorimotor neuropathy. (B) Schematic model of GSK3ß‐mediated neural development in SPG11‐ and CTRL‐NPCs. Increased GSK3 activity leads to reduced ß‐Catenin level in SPG11‐NPCs, thereby compromising proliferation and neurogenesis in SPG11 patients. Pharmacological treatment with the GSK3 inhibitors, tideglusib and CHIR99021, activates the canonical Wnt pathway by inhibiting GSK3 signaling and thereby restores proliferation and neurogenesis (shown by dashed green arrows). NPCs = cortical neural progenitor cells.