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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Neurobiol Aging. 2020 Feb 19;90:125–134. doi: 10.1016/j.neurobiolaging.2020.02.011

A three-dimensional dementia model reveals spontaneous cell cycle reentry and a senescence-associated secretory phenotype

Veronica Porterfield 1,2,3, Shahzad S Khan 4, Erin P Foff 1, Mehmet Murat Koseoglu 4,5,6, Isabella K Blanco 5, Sruthi Jayaraman 5, Eric Lien 5, Michael J McConnell 7,8, George S Bloom 3,4,8, John S Lazo 5,6, Elizabeth R Sharlow 5,6
PMCID: PMC7166179  NIHMSID: NIHMS1563589  PMID: 32184029

Abstract

A hexanucleotide repeat expansion on chromosome 9 open reading frame 72 (C9orf72) is associated with familial Amyotrophic lateral sclerosis (ALS) and a subpopulation of patients with sporadic ALS and frontotemporal dementia (FTD). We used inducible pluripotent stem cells from neurotypic and C9orf72+ (C9+) ALS patients to derive neuronal progenitor cells (NPCs). We demonstrated that C9+ and neurotypic NPCs differentiate into neurons. The C9+ neurons, however, spontaneously reexpressed cyclin D1 after 12 weeks, suggesting cell cycle reengagement. Gene profiling revealed significant increases in senescence associated genes in C9+ neurons. Moreover, C9+ neurons expressed high levels of mRNA for CXCL8, a chemokine overexpressed by senescent cells, while media from C9+ neurons contained significant levels of CXCL8, CXCL1, IL13, IP10, CX3CL1 and reactive oxygen species, which are components of the senescence-associated secretory phenotype. Thus, reengagement of cell cycle-associated proteins and a senescence-associated secretory phenotype, could be fundamental components of neuronal dysfunction in ALS and FTD.

Keywords: frontotemporal dementia, amyotrophic lateral sclerosis, cell cycle reentry, senescence, senescence-associated secretory phenotype

1. Introduction

Amyotrophic lateral sclerosis (ALS) is a disease that primarily targets motor neurons resulting in their degeneration and death. Approximately 16,000 people in the United States have ALS and ~5,000 new cases are diagnosed each year (Mehta et al., 2016; Wagner et al., 2015). ALS patients can exhibit cognitive impairment, behavioral issues, and/or dementia, and ALS exhibits significant genetic and functional overlap with the neurodegenerative disorder, Frontotemporal dementia (FTD) (Lattante et al., 2015). FTD is the second most common presenile dementia after Alzheimer’s disease, with manifestations including behavioral disturbances, disinhibition, cognitive decline and/or language dysfunction (Lattante et al., 2015). Ten to fifteen percent of FTD patients have a family history of FTD (Takada, 2015) and up to 40% of FTD patients have concomitant motor neuron disease or motor system dysfunction (Burrell et al., 2011). A hexanucleotide repeat expansion (GGGGCC) on chromosome 9 open reading frame 72 (C9orf72) has been associated with familial ALS and a segment of sporadic ALS and FTD patients (DeJesus-Hernandez et al., 2011). C9orf72 repeat expansion has also been identified as an atypical cause of other neurodegenerative disorders including Parkinson’s, Creutzfeldt-Jakob and Alzheimer’s diseases (Balendra and Isaacs, 2018), likely reflecting the phenotypic heterogeneity complicating clinic-pathological correlation of neurodegenerative diseases and indicating the pleiotropic nature of C9orf72 expansion in neural death.

In neurotypic individuals, there usually are ≤11 hexanucleotide repeats within the C9orf72 gene (Balendra and Isaacs, 2018; Harms et al., 2013; Rutherford et al., 2012; van der Zee et al., 2013). In contrast, in C9+ALS/FTD patients generally have >30 hexanucleotide repeats; however, some C9+ ALS/FTD patients exhibit expansions ranging from hundreds to thousands of repeats. Critically, these hexanucleotide repeats are transcribed into RNA and can be translated into dipeptide repeat proteins (DPRs) (Ash et al., 2013; Balendra and Isaacs, 2018; Donnelly et al., 2013; Gendron et al., 2013; Lagier-Tourenne et al., 2013; Mizielinska et al., 2013; Mori et al., 2013a; Mori et al., 2013b; Zu et al., 2013). Three main competing mechanisms have been posited for the pathogenic effects of C9orf72: a) loss of C9orf72 protein, b) toxic gain of function from C9orf72 repeat RNA or c) toxic gain of function from DPRs (Balendra and Isaacs, 2018). Recently, patient-derived stem cell models have proven useful in recapitulating several key in vivo pathological features of C9orf72 ALS/FTD. These include: a) RNA foci comprising both the sense and antisense direction repeat-laden mRNA (DeJesus-Hernandez et al., 2011); b) the formation of repeat-associated non-AUG translation (RAN) dipeptides (Ash et al., 2013; Mori et al., 2013b; Wen et al., 2014); c) disruptions in nuclear-cytoplasmic transport (both import and export) (Freibaum et al., 2015; Zhang et al., 2015); and d) glutamate toxicity compared to non-ALS iPS neurons (Zhang et al., 2015).

Most studies on C9orf72 ALS/FTD have focused on motor neurons, while only a few have centered on cortical neurons (Guo et al., 2017). Given that ALS patients can present with FTD symptoms (Ling et al., 2013; Ng et al., 2015) and C9orf72 has been found in other neurodegenerative disorders, such as Alzheimer’s disease, we wanted to establish a relevant cell culture model that more accurately reflected the areas of the ALS/FTD brain affected by neurodegenerative processes. We hypothesized that an in vitro three-dimensional culture system derived from cortical neural progenitor cells (NPCs) generated from C9orf72 ALS patients would provide a powerful in vitro model for ALS/FTD, one which more closely mimics native brain morphology and pathology than standard two-dimensional cultures. Therefore, we developed and interrogated such a system, and uncovered spontaneous ectopic cell cycle re-entry and concomitant expression of soluble factors linked with the senescence-associated secretory phenotype in the C9+ iPS neurons, suggesting a novel mechanism for neuronal dysfunction in ALS/FTD.

2. Materials and Methods

2.1. Expansion of inducible pluripotent stem cells (iPSC) and creation of neural progenitor cells.

The following C9+ iPSC lines, CS29iALS-C9n1N (29i) and CS30iALS-C9n1A (30i), were obtained from the Cedars-Sinai Medical Center’s David and Janet Polak Foundation Stem Cell Core Laboratory (Los Angeles, CA) (Supplemental Table 1). Protocols for expanding the C9+ iPSCs were provided by the Cedar-Sinai Medical Center’s David and Janet Polak Foundation Stem Cell Core Laboratory. Briefly, colonies were grown in mTESR™1 medium (Stem Cell Technologies, Cambridge, MA) and passaged until the generation of embryoid bodies (EB). Once at EB stage, all lines were handled as previously described (Brennand et al., 2011) with the exception that neural rosettes and progenitor cells were derived using DMEM/F12+Glutamax medium (ThermoFisher, Waltham, MA) supplemented with 1% N2 (ThermoFisher), 2% B27 without vitamin A (ThermoFisher), 20 μg/mL fibroblast growth factor 2 (FGF2; Peprotech, Rocky Hill, NJ) and 1 μg/mL laminin (Invitrogen, Carlsbad, CA) and were cultured on Matrigel™ (Corning, Corning, NY) coated six-well tissue culture plates. NPCs were stored in liquid nitrogen until use (<12 months). The identity of NPC cultures was confirmed by immunofluorescence (IFC) for Nestin and Sox2, with few cells positive for doublecortin and MAP2.

2.2. NPCs.

The neurotypic control NPC lines, BOH1 and 9319a, were derived from iPSC lines obtained from Dr. K. Brennand (Icahn School of Medicine at Mount Sinai, New York, NY) (Supplemental Table 1). The neurotypic NPC control line, 7545–5b, was derived from cell line GM07545 from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ) and obtained from the University of Virginia Stem Cell Core (Charlottesville, VA) (Supplemental Table 1). All neurotypic NPC control lines were grown and passaged as previously described (Kim et al., 2011).

2.3. NPC differentiation into neurons.

NPCs were thawed and expanded on Matrigel™-coated dishes in NPC medium and split approximately 1:4 every week (up to passage 15) using Accutase (MilliporeSigma, St. Louis, MO). For 2D IFC studies, cells (i.e., passage 10) were seeded at a density of 25 cells/μL onto poly-D-ornithine/laminin-coated coverslips. At 48 hr post-seeding, the culture medium was aspirated and neuron differentiation medium (DMEM/F12+Glutamax, 1% N2, 2% B27 with vitamin A, 20 ng/mL BDNF (Shenandoah Biotechnology, Warwick, PA), 20 ng/mL GDNF (Shenandoah Biotechnology), 500 μM dibutyryl cyclic AMP (MilliporeSigma), 200 nM ascorbic acid (MilliporeSigma), and 1% penicillin-streptomycin (ThermoFisher)) was then added to promote cortical neuronal differentiation. For the first week, differentiation medium was replaced two times. After week one, neuronal differentiation medium supplemented with 1 μg/mL human laminin (Invitrogen) was used as maintenance medium and was replaced at least once per week.

For 3D cultures, Alvetex (ALV) 12-well inserts (ReproCell, Glasgow, UK) were placed into 12-well tissue cell culture plates. Scaffolds were prepared for cell seeding according to manufacturer’s instructions and then coated with 500 μL of human laminin (10 μg/mL) (Invitrogen) for one hr at room temperature (RT). Excess laminin was aspirated from each well. NPCs (i.e., passage 10) were then plated onto each scaffold at a density of 6.28 × 103 cells/μL in 75 μL unsupplemented DMEM/F12+Glutamax medium and allowed to settle into the scaffold for one hr at 37°C. Four mL of NPC medium were then added to each scaffolded well and cultures were incubated for 24 hr. Neuronal differentiation was initiated as described above.

2.4. Cell viability assay.

Calcein AM (CAM) (ThermoFisher) and propidium iodide (PI) (ThermoFisher) were used to assess neuron viability at 8–9 and 43–44 days using both 2D and 3D culturing conditions. For each cell culture condition, three wells were rinsed gently in 37°C Dulbecco’s Phosphate Buffered Saline (DPBS) and then treated with one μM CAM-PI solution in pre-warmed DPBS. Cells were incubated for 30 min at 37°C. CAM-PI was then aspirated and replaced with DPBS at 37°C. Cells were incubated at 37°C for 30 min prior to capturing the images. Three to four images were obtained per culturing condition were taken on a Zeiss 710 Multiphoton Confocal microscope (University of Virginia Advanced Microscopy Core) with a 20x water immersion lens (NA=1.0). Cell counts for the number of live-dead cells were derived using ImageJ (Rueden et al., 2017). A total of 800–1000 cells were counted per replicate per culturing condition.

2.5. Fluorescence immunocytochemistry.

Cells were fixed for 30 min (2D and ALV scaffold) at RT in 4% paraformaldehyde (PFA). Cells were washed three times for 20 min in PBS with 0.1% Tween 20 (PBST). Blocking and permeabilization occurred simultaneously in a solution containing IFC block (2% BSA (Rockland Immunochemicals, Pottstown, PA), 1% fish skin gelatin (MilliporeSigma), 0.02% saponin (Alfa Aesar, Haverhill, MA), 15% horse serum (ThermoFisher) in PBS and 0.3% Triton X-100 (MilliporeSigma) for 4 hr at RT. Primary antibodies (Supplemental Table 2) were diluted in IFC block without Triton X-100, added to permeabilized cells and incubated overnight at 4°C. After incubation, cells were washed twice for 15 min in PBST at RT followed by three washes for 30 min also using PBST at RT. All AlexaFluor (ThermoFisher) secondary antibodies (Supplemental Table 2) were diluted to 2 μg/mL in IF block and incubated for 4 hr at RT. Cells were washed as described above; however, the final wash was with deionized, distilled water (ddH20). Cells were then exposed to DAPI (ThermoFisher, 1:1000 in ddH20) for 10 min at RT, washed three times for 10 min in ddH20 and then mounted on slides with Mowiol (MilliporeSigma). To remove bias towards neuronal markers, we first found scaffolded and 2D fields of view by locating areas of DAPI stained nuclei, and then used other filters to image MAP2, BIII tubulin, tyrosine hydroxylase (TH), and cyclin D1. Images were captured on a Zeiss 710 Multiphoton Confocal microscope as described earlier for the cell viability assay. Cell counts for the number of immunopositive cells are reported as the ratio of immunopositive cells to the total number of DAPI nuclei in an image. Cells counts were derived using ImageJ (Rueden et al., 2017). A total of 150–1500 cells were counted per replicate per culturing condition.

2.6. RNA purification, cDNA synthesis and qPCR.

RNA was purified from each culturing condition as follows. For 2D cell cultures, cells were dissociated with Accutase for 5 min at 37°C, then centrifuged at 1000 × g for 5 min at RT. Cell pellets were washed twice with DPBS. RNA from 2D cell pellets was isolated using an RNeasy kit following the manufacturer’s instructions (Qiagen, Germantown, MD) or flash frozen in liquid nitrogen and stored at −80°C until RNA isolation. For ALV cultures, scaffolds with attached cells were removed from the hanging inserts, placed into a new 12-well plate and washed twice with PBS. Cells were directly lysed with 350 μL RLT lysis buffer with β-mercaptoethanol (1%) for 10 min per manufacturer’s instructions. Cell lysates were passed through a 20-gauge syringe 20 times and then loaded onto a column. The RNeasy protocol was then followed. Real time quantitative PCR was performed with a BioRad CFX 96 using 20 μL duplicate reactions that were made using BioRad SsoAdvanced SYBR green with BIO RAD 20x Primer Assays. mRNA for the housekeeping genes, β-ACTIN, HPRT and/or GAPDH, were used to normalize and calculate ΔΔCTs for gene expression.

Human cell cycle RT2-PCR microarrays were purchased from Qiagen. Total RNA from neurotypic and the C9+ 29i and 30i iPS neurons was purified using an RNeasy Plus RNA isolation kit (Qiagen) per the manufacturer’s instructions. A total of 500–1000 ng of mRNA were converted to cDNA using the RT2 first strand synthesis kit (Qiagen). RT2-PCR microarrays were processed using manufacturer’s instructions. Real-time monitoring of the qPCR reaction was performed on a BioRad CFX Connect thermocycler with RT2 SYBR Green ROX Mastermix (Qiagen). The program was run at 95°C for 10 min and 40 cycles at 95°C for 15 sec and 60°C for one min. RT2-PCR microarray data were analyzed using the Qiagen data webportal (https://www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/). Gene expression was normalized against internal PCR microarray controls (i.e., β-ACTIN, HPRT and GAPDH) using the geometric mean of replicate samples. Significant gene expression was determined by Z-score >1.5. Each cell line under each culture condition was subject to three independent microarray assessments and derived data were used to create volcano plots.

Total RNA was purified from human ALS and ALS/FTD patient brain samples (kind gift from Drs. Timothy Miller and Matthew Harms, Department of Neurology, Washington University in St. Louis, MO) using an RNeasy Plus RNA (Qiagen) isolation kit per the manufacturer’s protocol (Supplemental Table 3). qPCR was performed as described above.

2.7. Luminex analysis of cell culture supernatants derived from iPS neurons cultured on ALV scaffolds.

The quantitative analysis of 41 human chemokines and cytokines was performed by the University of Virginia Flow Cytometry Core Facility. ALV scaffolds were prepared and seeded with neurotypic NPCS (i.e., 7545), 29i or 30i iPS NPCs and differentiated for 12 weeks as described above. At 12 weeks, media from cells were harvested, precleared by centrifugation at 14,000 × g at 4°C, and stored at −80°C until Luminex analysis. Cell supernatants were analyzed for the presence of 41 different cytokines and chemokines using a MILLIPLEX MAP human cytokine/chemokine premixed panel (EMDMillipore, Billerica, MA) using a Luminex MAGPIX system (Luminex, Austin, TX). The analytes were as follows: EGF, eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF, GM-CSF, GRO, IFNα2, IFN-γ, IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40 and p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, TNF-α, TGF-α, TNF-β, and VEGF.

2.8. Assessment of reactive oxygen species generation.

Cell media supernatants, isolated and prepared from ALV scaffolds as described above, were evaluated for reactive oxygen species (ROS) using ROS-Glo (Promega, Madison, WI) according to manufacturer’s instructions.

2.9. Statistical analyses.

All statistical analyses, including the Benjamini-Hochberg test, were performed with GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). Data are presented as average (mean) ± SD or SEM. p values were calculated with Student’s t test for comparisons involving two groups or one-way or two-way ANOVA for comparisons involving >two groups. p<0.05 was considered statistically significant. Each experiment is represented by at least three biological replicates and three technical replicates (per independent experiment).

3.0. Reactome analysis.

A Reactome analysis was performed by accessing the https://reactome.org webportal. Common up-regulated and down-regulated genes from the C9+ 29i and 30i versus BOH1 RT2-PCR microarrays were used for the analyses. Pathways most affected by the gene expression patterns were identified and stratified by significance, p-value and false discovery rate (FDR).

3. Results

3.1. C9orf72 (C9+) ALS/FTD iPS cortical neurons express similar neuronal markers as neurotypic controls.

We created iPS NPCs from the patient-derived C9orf72+ iPS cells lines, CS29iALS-C9n1N and CS30iALS-C9n1A, as previously described (Brennand et al., 2011). We fated the NPCs to cortical neurons, the cells most affected in FTD. The derived CS29iALS-C9n1N (29i) and CS30iALS-C9n1A (30i) (i.e., C9+) NPC lines formed neurons using both traditional (i.e., 2D cell culture plastic) and 3D (i.e., ALV scaffolds) cell culture methods. Quantitative PCR analyses at 11 days and 6 weeks post-differentiation showed MAP2 mRNA expression in C9+ 2D and ALV 3D cultures (Figure 1a). Immunofluorescence studies identified MAP2+ neurons in the 29i and 30i 2D and ALV cell models at similar frequencies (Figure 1b, c) and corresponding to levels expressed by neurotypic NPCs by 6 weeks. βIII tubulin and tyrosine hydroxylase expression were also detected at 3 and 6-weeks post-differentiation (Supplemental Figure 1a–b). These data suggested that C9+ iPS neurons displayed similar neuronal differentiation patterns and neuronal markers as neurotypic iPS neurons.

Figure 1. C9+ iPS neurons express neuronal markers in 2D and 3D culture.

Figure 1.

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Panel a, Map2 gene expression levels by qPCR in neurotypic (i.e., BOH1 and 9319a) and C9+ (i.e., 29i and 30i) iPS neurons. Black bars, 11 days post-differentiation induction. White bars, 6 weeks post-differentiation induction. (--- undifferentiated control). N=2 per cell line. Panel b, MAP2 percent positive cells as detected by immunofluorescence at 6 weeks post-differentiation induction. Panel c, Neurotypic (BOH1) and C9+ iPS neurons after differentiation on ALV scaffolding for 6 weeks. Neurons (MAP2, green), astrocytes (GFAP, pink) and nuclei (DAPI, blue), (20x objective, NA 0.8, scale bar 100μm). Panel d, Viability of C9+ ALS/FTD and neurotypic neurons at 8 days (black bars) and 6 weeks (white bars) post-differentiation induction using calcein AM. N=12 per cell line.

In 2D, neurotypic and C9+ neuronal cultures displayed similar levels of viability at 8 days and 6 weeks (Figure 1d). Noteworthy, however, was the statistically significant decrease in the viability of the 3D C9+ 30i iPS neuronal co-cultures at 8 days and 6 weeks differentiation (Figure 1d). This viability decrease was not observed in the C9+ 29i cultures (Figure 1d). However, with extended culturing in 2D and 3D (>200 days), both C9+ cell lines trended with higher mortality versus neurotypic cells (Supplemental Figure 1d). These data suggested that, although the C9+ iPS neuronal cultures have similar differentiation patterns, they may be characterized by a general loss of fitness versus neurotypic controls. However, we note that the calcein AM viability assessment provides insight to the health of the overall neuronal co-culture and precise cell populations affected are not discernible.

3.2. C9+ iPS neurons spontaneously express cyclin D1 after 12 weeks in 3D culture.

Control neurotypic and C9+ NPCs were seeded in 2D as well as in ALV scaffolds and then differentiated into neurons for up to 3 months. 2D cultures and ALV scaffolds with differentiating neurotypic or C9+ NPCs were then immunostained for expression of cyclin D1, a marker of G1 of the cell cycle, at 6 and 12 weeks after induction of differentiation (Figure 2). Immunostaining of the ALV scaffolds for cyclin D1 protein levels revealed a statistically significant increase in the C9+ versus neurotypic iPS neuronal cultures at 12 weeks post-differentiation (Figure 2b). We conclude that C9+ neurons grown in 3D culture spontaneously re-enter the cell cycle, which interestingly precedes most neuron death in Alzheimer’s disease. In contrast, no such statistically significant difference in cyclin D1 expression for C9+ versus neurotypic iPS neurons was observed in 2D cultures at any time point (Figure 2c); however, the data suggested a similar trend in cyclin D1 expression upon extended culturing.

Figure 2. ALS/FTD C9+ cell lines spontaneously reenter the cell cycle.

Figure 2.

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Panel a. ALS/FTD C9+ (29i and 30i) and neurotypic cell lines spontaneously express cyclin D1 protein in 3D culture 12-weeks post-differentiation. Shown are representative images of the 9319a and 29i+ cell lines. ALV cultures were stained for IF with anti-MAP2 (green) and anti-cyclin D1 (CYD1, red), which identifies mature neurons and cells that have exited G0 and entered into G1, respectively. Nuclei were stained with DAPI (blue) (20x objective, NA 0.8, scale bar 100μm). Panel b. Quantification of cyclin D1 expression in neurotypic (black bars) and C9+ (white bars) cells grown in 2D and on ALV scaffolding. N=3 (9319a, 29i,) and N=2 (BOH1, 30i) at 6 weeks a minimum of 800 cells was counted total across each replicate; N=2 (9319a, BOH1, 29i and 30i) at 12 weeks; a minimum of 150 cells was counted across each replicate.

3.3. C9+-derived iPS neuronal cultures have dysregulated cell cycle-associated gene expression.

To characterize more fully the observed spontaneous re-expression of cyclin D1 by C9+ iPS neurons at 12 weeks in 3D culture, we harvested RNA from 29i and 30i C9+ and neurotypic (BOH1) iPS neuron cultures at 3-months post-differentiation and performed a cell cycle gene-targeted RT2-PCR array, which comprised 84 genes associated with the mammalian cell cycle. In the 29i C9+ iPS neurons, the expression levels of four genes were upregulated (fold change>1.5) under basal 3D culturing conditions: CDKN2A, CDKN2B, CDKN1A and CCND1 and three genes were downregulated in expression: AURKB, GTSE1 and CCNA2 (Figure 3a). In the 30i C9+ iPS neurons, in contrast, four genes were upregulated (i.e., CDKN2A, CDKN2B, CCNH, and CCND3), while ten genes were downregulated (i.e., AURKB, CDK2, MCM3, KPNA2, MKI67, ATM, CCNB1, CDC20, GTSE1, and CDK1) (fold change >1.5) (Figure 3b).

Figure 3. Volcano plot of relative differences in mRNA gene expression of two C9+ ALS versus neurotypic cells cultured for three months on ALV scaffolding.

Figure 3.

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Genes significantly overexpressed (fold change >1.5 or <−1.5) are in red (•) with remaining genes being denoted in green (•). The dashed line = p<0.05. N=3 independent microarrays per cell line. BOH1 cell line used as the neurotypic control.

We next confirmed the gene expression of the overlapping gene set (i.e., CDKN2A, CDKN2B, AURKB and GTSE1) (Figure 4) using RNA isolated from 29i C9+ iPS neurons cultured for 3 months on ALV. Benjamini-Hochberg analysis predicted that CDKN2A, CDKN2B and AURKB were true positives while GTSE1 might be a false positive (Supplemental Table 4). Using qRT-PCR, we confirmed CDKN2A and CDKN2B gene expression was upregulated ~20- and ~5-fold, respectively, versus neurotypic control (Figure 4a–b). AURKB downregulation was confirmed in both 29i iPS neuron RNA samples (Figure 4c), while downregulation of GTSE1 gene expression was confirmed in only one of the two 29i iPS neuron RNA samples (Figure 4d) supporting the computational Benjamini-Hochberg analysis (Supplemental Table 4) which suggested it may be a false positive.

Figure 4. qPCR confirmation of CDKN2A, CDKN2B, GTSE1 and AURKB gene expression in C9+ 29i ALS cell line versus BOH1 neurotypic control cell line.

Figure 4.

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mRNA gene expression levels were normalized relative to ACTIN, HPRT and GAPDH. N=2 biological replicates. N=3 technical replicates per control gene ± SEM. Panel a, CDKN2A. Panel b, CDKN2B. Panel c, GTSE1. Panel d, AURKB. A, replicate 1. B, replicate 2.

3.4. Computational analysis of overlapping gene sets suggest cellular senescence pathways are activated in 29i and 30i C9+ neurons.

To identify possible cellular pathways most affected by the CDKN2A, CDKN2B, and AURKB gene signature in the C9+ iPS neurons, we performed computational analysis using Reactome (Fabregat et al., 2016), which suggested that the cellular responses to stress pathway node was significantly over-represented in the C9+ iPS neurons based on the CDKN2A, CDKN2B and AURKB gene signature (Figure 5a, Supplemental Figure 1). Moreover, there was a specific emphasis on pathways leading to overall cellular senescence, including oncogene induced senescence, oxidative stress induced senescence, cellular senescence and the senescence-associated secretory phenotype (Figure 5a, Table 1).

Figure 5. Reactome analysis of overlapping gene hits from 29i and 30i ALS/FTD C9+ cell lines.

Figure 5.

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Panel a, Reactome (https://reactome.org) computational analysis of the common gene hits from the qRT-PCR microarrays highlight the potential involvement of cellular senescence pathways in the CCR of the 29i and 30i cell lines after 12 weeks in 3D culture. Panel b, qPCR was performed on RNA from 29i, 30i and 7545 neurotypic control cells to quantify CXCL8 gene expression. Panel c, Luminex analysis was performed for detection of secreted CXCL8. N=3 replicates ± SEM. Error bars=SEM.

Table 1.

Reactome analyses

PATHWAY NAME p-value FDR
Oncogene induced senescence 8.33E-7 1.52E-4
Oxidative stress induced senescence 2.57E-5 2.34E-3
Cellular senescence 1.14E-4 6.95E-3
Senescence-associated secretory phenotype 2.47E-3 1.11E-1
Cellular response to stress 5.34E-3 1.92E-1
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3.5. ALD/FTD C9+ iPS neurons express components of the senescence-associated secretory phenotype.

To confirm that the 29i and 30i C9+ ALS/FTD iPS neuronal cultures were indeed undergoing senescence at 12-weeks in 3D culture, we performed qPCR on 29i and 30i iPS neuron-derived RNA for detection of IL-6 and/or CXCL8 (IL-8), which are two soluble factors closely associated with cellular senescence (Tan et al., 2014). CXCL8 gene expression was significantly upregulated in 29i and 30i C9+ iPS neurons (Figure 5b) compared to neurotypic controls, while there was no detectable change in IL-6 gene expression (data not shown). Nonetheless, the detection of upregulated CXCL8 gene expression suggested that the C9+ iPS neurons were indeed undergoing senescence. Subsequently, we initiated additional 29i, 30i and neurotypic iPS neuronal cultures in 3D, maintained them in culture for 12 weeks, and then harvested the cell culture media for quantification of secreted cytokines and chemokines by bead-based multianalyte profiling. We confirmed the significant overexpression of components of the senescence-associated secretory phenotype including CXCL8 (IL-8), CCL2 (MCP1), CXCL1 (GROα), IL-13 and IP10 (Figure 5c and Figure 6) in both the 29i and 30i ALS/FTD cell lines versus neurotypic controls. Additionally, CX3CL1 (fractalkine) was also significantly overexpressed in the C9+ ALS/FTD iPS neurons (Figure 6c). Fractalkine has been described as a “find me” (Sokolowski et al., 2014) signal for neurons undergoing ethanol-induced or glutamate-induced (i.e., excito-neurotoxicity) apoptosis as a means to promote efferocytosis through the recruitment of microglia (Noda et al., 2011; Sokolowski et al., 2014). We also detected in the media from 29i and 30i iPS neurons significantly elevated levels of ROS, which are considered inducers of cellular senescence (Tchkonia et al., 2013) (Figure 6f).

Figure 6. Detection of components of the senescence-associated secretory phenotype in cell culture supernatants from ALS/FTD C9+ cell lines.

Figure 6.

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29i, 30i and 7545 neurotypic control cells were seeded into ALV scaffolds and cultured for 12 weeks. Cell culture supernatants were harvested and assessed by Luminex analysis or for ROS detection. Panel a, CCL2 (MCP1). Panel b, CXCL1 (GROα). Panel c, CX3CL1 (fractalkine). Panel d, IL-13. Panel e, IP-10. Panel f, reactive oxygen species. Panel g, qPCR confirmation of CDKN2A and CDKN2B gene expression changes in human patient samples. ●, C9+. ●, C9−. mRNA levels were normalized relative to GAPDH. N=3 replicates ± SEM. Error bars=SEM. C=normal human brain RNA.

3.6. ALS/FTD patients have upregulated CDKN2A gene expression.

We next examined the gene expression levels of CDKN2A and CDKN2B in ALS and ALS/FTD patient brain RNA samples using qRT-PCR. CDKN2A gene expression was increased on average more than 4-fold in brain samples of patient with ALS and ALS/FTD (Figure 6g), while CDKN2B was unaltered. The range observed in CDKN2A expression levels may be in part dependent upon the time of post-mortem sampling (Supplemental Table 3). However, it would be more expected for RNA degradation to be observed, with extended post-mortem sampling periods, versus increased gene expression.

4. Discussion

The need for more pathologically relevant neurodegeneration models has become obvious with the growing realization of the limitations of mouse and traditional 2D cell culture systems (Centeno et al., 2018; Dawson et al., 2018; Jucker, 2010). Human iPSCs offer more physiologically relevant cell-based populations for experimental studies and can be differentiated into many cell lineages, such as cortical and motor neurons. Critically, patient-derived stem cell models have proven useful in demonstrating several key pathological features of C9orf72 ALS/FTD including: 1) RNA foci comprising both the sense and antisense direction repeat-laden mRNA (DeJesus-Hernandez et al., 2011); 2) formation of repeat-associated non-AUG translation (RAN) dipeptides (Ash et al., 2013; Mori et al., 2013b; Wen et al., 2014); 3) disruption of both import and export for nuclear-cytoplasmic transport (Freibaum et al., 2015; Zhang et al., 2015); and 4) glutamate toxicity compared to non-ALS iPS neurons (Donnelly et al., 2013). In general, however, the impact of iPS cells is most readily observable when cultured in 3D, which allows for the interactions with other cell populations. To that end, several groups have developed 3D iPS neuronal culture platforms (Hopkins et al., 2015; O’Connor et al., 2001; Yoo et al., 2011). Most notably, Choi et al. (Choi et al., 2014), cultured ReN cells in 3D and successfully replicated the Aβ and tau pathologies, as well as the inflammatory microenvironment (Park et al., 2018) found in Alzheimer’s disease, which is not discernible in 2D culture. Here, we demonstrate that differentiating C9+ iPS cells into cortical neurons in 3D is feasible and may help to further phenotyping of C9+ ALS/FTD. Furthermore, our 3D ALS/FTD C9+ iPS neuron-based model system recapitulates the reactivation of cell cycle regulators (i.e., cyclin D1) frequently observed in both neurodegeneration patients and corresponding in vitro models (Di Giovanni et al., 2005; Khan et al., 2018; Kodis et al., 2018; Lim and Qi, 2003; Maccioni et al., 2001; Norambuena et al., 2017; Pelegri et al., 2008; Ranganathan and Bowser, 2003, 2010; Rashidian et al., 2005; Rashidian et al., 2007; Seward et al., 2013).

Unfortunately, in neurodegenerative diseases, the neuronal loss of fitness and death occurs over the span of many years making the specific mode of neuron “loss” (e.g., apoptosis, necrosis, excitotoxicity, excess autophagy, synaptic loss, long-term potentiation impairment, neurite dysfunction, senescence), and any of its originating drivers, challenging to delineate. However, re-expression of cell cycle regulators (e.g., cyclin D1, CDK4, hyperphosphorylated pRb and E2F1) may be a common conduit leading to in vitro and in vivo neuronal dysfunction and death prior to and during neurodegenerative processes. There is significant evidence that neurons in vitro undergo cell cycle reentry in response to a variety of stressors, including neurotrophic factor deprivation (brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor), DNA damage, and oxidative stress (reviewed in (Zhu et al., 2004)). Neurons exposed to stressors usually die at the G1/S checkpoint prior to DNA synthesis; however, in some instances DNA synthesis as visualized by bromodeoxyuridine incorporation has been observed (Seward et al., 2013; Varvel et al., 2008). Recent evidence, however, suggests primary neurons can enter M-phase (albeit with an engineered neuronal cell system) with a small number of neurons actually dividing (Walton et al., 2019), although it is likely neurons more commonly exist in a prolonged 4N state (Frade and Ovejero-Benito, 2015). Nonetheless, these data suggest that neurons retain the cell cycle machinery needed for cell cycle progression (Walton et al., 2019), and with the appropriate stimuli, neurons have the capacity to re-engage the cell cycle.

Neurons may be particularly sensitive to oxidative stress. The “two-hit” model of neurodegeneration (Zhu et al., 2004) suggests that oxidative stress in conjunction with re-engagement of the cell cycle machinery in “at risk” neurons may be necessary for neurodegenerative progression. In this model, cell cycle re-entry and elevated oxidative stress can exist separately (i.e., “single hit”), but when experienced concurrently (“second hit”), the neurodegenerative process initiates (Zhu et al., 2004). Moreover, this model proposes the existence of an “oxidative steady-state” in which “at risk” neurons are chronically bombarded with ROS yet remain functional for years. These neurons are thought to be exquisitely sensitive to the “second hit”, which provides the impetus toward neurodegeneration (LeBel and Bondy, 1992; Zhu et al., 2004). Oxidative stress also triggers cellular senescence, which is defined as irreversible or sustained cell cycle arrest; however, senescence is a complex cellular response to a variety of stressors, including mitochondrial deterioration, ROS, and oncogene expression (Campisi, 2013). Senescence also promotes chronic inflammation, a state referred as “inflammaging” (Franceschi et al., 2018; Kritsilis et al., 2018), which is caused by increased secretion of pro-inflammatory cytokines and other soluble factors. These proinflammatory cytokines often engage signaling effectors associated with the cell cycle or that serve to regulate the expression of cell cycle proteins and may deliver the “second hit” required for neurodegenerative progression (Cingoz and Goff, 2018; Leslie et al., 2006; Mori et al., 2013a; Tian et al., 2017). Notably, we detected multiple proinflammatory chemokines (i.e., CXCL8, CCL2, CXCL1, and IP-10) in our 3D ALS/FTD neuronal cultures as part of the senescence-associated secretory phenotype. Moreover, IL-13, which was also detected in our ALS/FTD neuronal cultures, can sensitize cells to oxidative stress during neuro-inflammatory conditions (reviewed in (Mori et al., 2016)).

Unfortunately, universally accepted biochemical markers of senescence are not yet available, but examples of senescence markers that have been used include p16INK4a and p21 expression, β-galactosidase activity, telomere-associated foci, epigenomic alterations, ROS, DNA damage and the senescence–associated secretory phenotype (SASP) (Myrianthopoulos et al., 2019; Sun et al., 2018). Some frequently used endpoints, such as β-galactosidase activity, have not been useful with neurons (Piechota et al., 2016). Although most commonly associated with cancer and aging, cellular senescence may contribute to, and perhaps drive, neurodegenerative diseases. Several recent studies have demonstrated that senescent cells (i.e., astrocytes, microglia, oligodendrocytes, oligodendrocyte progenitor cells, neurons, and neural stem cells) (reviewed in (Kritsilis et al., 2018)) exist within the brains of aging organisms, as well as in brains with neurodegenerative disease, suggesting that they promote neuronal dysfunction (Musi et al., 2018; Oost et al., 2018) and the associated cognitive decline (Bussian et al., 2018).

If either senescence or cell cycle re-entry are drivers of ALS/FTD pathology, then it is conceivable that compounds that target these processes, such as the senolytic (Myrianthopoulos et al., 2019; Xu et al., 2018) or cell cycle inhibitors (Xu et al., 2013), might have some prophylactic or therapeutic potential for the disease. The 3D iPSC model, which we have developed, might be a valuable tool to help facilitate these studies especially since it comprises neuronal as well as glial cell populations. Thus, in vitro and in vivo models that recapitulate these cell cycle re-entry SASP phenotypes may be invaluable resources to understand the timing and progression of ALS/FTD associated neurodegenerative events.

Supplementary Material

Supplemental Figures and Tables

Supplemental Figure 1. Panels a and b, Expression of βIII tubulin and tyrosine hydroxylase (TH) by neurotypic 9319a and C9+ 30i iPS neurons. Panel c, Alexa fluor secondary antibody only control. No primary antibodies were added. Anti-chicken IgY 488, anti-rabbit IgG 568, and anti-mouse IgG 647. Scale bar = 100 μm. Panel d, Viability assessment of neurotypic BOH1, C9+ 29i and C9+ 30i iPS neuronal cultures at 200 days post-differentiation.

Supplemental Figure 2. Venn diagram of differentially expressed cell cycle-associated genes in 29i and 30i iPS neurons.

Supplemental Table 1. C9+ cell lines used for neuronal progenitor cell derivation.

Supplemental Table 2. Antibodies used in studies.

Supplemental Table 3. Patient information.

Supplemental Table 4

Supplemental Table 4. Benjamini-Hochberg test on C9+ 29i and 30i p-values. Red text indicates hits from C9+ 29i PCR microarray studies. Bolded red text indicates genes that confirmed in C9+ 29i confirmation studies. Bolded black text indicates additional hits from C9+ 30i PCR microarray studies.

Highlights.

  • Neurotypic and C9+ NPCs can be differentiated in two- and three-dimensional models.

  • C9+ NPCs spontaneously expressed cyclin D1 protein in three-dimensional culture.

  • Expression profiling showed increases in genes associated with cellular senescence.

  • C9+ neurons expressed factors of the senescence-associated secretory phenotype.

5. Acknowledgments

The work was supported by grants from the Fiske Drug Discovery Fund (J.S.L., E.R.S.), the Owens Foundation (E.F., J.S.L., G.S.B.), NIH RF1 AG51085 (G.S.B.), NIH R01 AG063400 (E.R.S.), Hartwell Foundation (E.F.) and the Cure Alzheimer’s Fund (G.S.B., J.S.L, E.R.S.). This work used the Zeiss 710 Confocal Microscope in the Advanced Microscopy Facility, the Luminex MagPix in the Flow Cytometry Core and neural progenitor cells produced by the Stem Cell Core Facility, which are all facilities supported by the University of Virginia School of Medicine. We also thank the Cedars-Sinai Medical Center’s David and Janet Polak Foundation Stem Cell Core Laboratory for the ALS C9+ iPS cells. This manuscript is dedicated to the memory of James F. Capps (1958-2015).

Footnotes

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6.

Disclosure statement

The authors have nothing to disclose. Dr. E. Foff now works for Acadia Pharmaceuticals. Dr. Shahzad Khan now works at Stanford University.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figures and Tables

Supplemental Figure 1. Panels a and b, Expression of βIII tubulin and tyrosine hydroxylase (TH) by neurotypic 9319a and C9+ 30i iPS neurons. Panel c, Alexa fluor secondary antibody only control. No primary antibodies were added. Anti-chicken IgY 488, anti-rabbit IgG 568, and anti-mouse IgG 647. Scale bar = 100 μm. Panel d, Viability assessment of neurotypic BOH1, C9+ 29i and C9+ 30i iPS neuronal cultures at 200 days post-differentiation.

Supplemental Figure 2. Venn diagram of differentially expressed cell cycle-associated genes in 29i and 30i iPS neurons.

Supplemental Table 1. C9+ cell lines used for neuronal progenitor cell derivation.

Supplemental Table 2. Antibodies used in studies.

Supplemental Table 3. Patient information.

NIHMS1563589-supplement-Supplemental_Figures_and_Tables.pdf (536.5KB, pdf)
Supplemental Table 4

Supplemental Table 4. Benjamini-Hochberg test on C9+ 29i and 30i p-values. Red text indicates hits from C9+ 29i PCR microarray studies. Bolded red text indicates genes that confirmed in C9+ 29i confirmation studies. Bolded black text indicates additional hits from C9+ 30i PCR microarray studies.

NIHMS1563589-supplement-Supplemental_Table_4.xlsx (17.6KB, xlsx)

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