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. Author manuscript; available in PMC: 2023 Oct 13.
Published in final edited form as: J Neurogenet. 2022 Oct 13;36(2-3):81–87. doi: 10.1080/01677063.2022.2129632

Abnormalities of Neural Stem Cells in Lesch-Nyhan Disease

Ashok R Dinasarapu 1, Diane J Sutcliffe 2, Fatemeh Seifar 2, Jasper E Visser 3,4, H A Jinnah 5
PMCID: PMC9847586  NIHMSID: NIHMS1862228  PMID: 36226509

Abstract

Lesch-Nyhan disease (LND) is a neurodevelopmental disorder caused by variants in the HPRT1 gene, which encodes the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt). HGprt deficiency provokes numerous metabolic changes which vary among different cell types, making it unclear which changes are most relevant for abnormal neural development. To begin to elucidate the consequences of HGprt deficiency for developing human neurons, neural stem cells (NSCs) were prepared from 6 induced pluripotent stem cell (iPSC) lines from individuals with LND and compared to 6 normal healthy controls. For all 12 lines, gene expression profiles were determined by RNA-seq and protein expression profiles were determined by shotgun proteomics. The LND lines revealed significant changes in expression of multiple genes and proteins. There was little overlap in findings between iPSCs and NSCs, confirming the impact of HGprt deficiency depends on cell type. For NSCs, gene expression studies pointed towards abnormalities in WNT signaling, which is known to play a role in neural development. Protein expression studies pointed to abnormalities in the mitochondrial F0F1 ATPase, which plays a role in maintaining cellular energy. These studies point to some mechanisms that may be responsible for abnormal neural development in LND.

Keywords: Lesch-Nyhan disease, HPRT1, purine salvage, development, induced pluripotent stem cell

Introduction

Lesch-Nyhan disease (LND) is an inherited metabolic disorder associated with characteristic neurobehavioral problems that emerge at an early age. LND is caused by defects in the HPRT1 gene, leading to deficiency of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt), which is involved in purine recycling. The mechanisms by which HGprt deficiency lead to the neurodevelopmental problems have yet to be determined.

Anatomical studies of human LND brains collected at autopsy have not revealed any overt structural malformations or signs of neurodegeneration (Goettle et al., 2014; Jinnah et al., 2006), although advanced imaging methods have shown subtle loss of volume in certain regions (Del Bene et al., 2021; Schretlen et al., 2013; Schretlen et al., 2015). The brains of adult HPRT1 knockout mice also show no obvious structural anomalies (Egami et al., 2007; Jinnah et al., 1994). Instead, several studies have pointed to abnormalities of early neural development such as reduced neurite growth in the HPRT1 knockout mice (Mikolaenko et al., 2005) or neuron-like tissue culture models of HGprt deficiency (Ceballos-Picot et al., 2009; Cristini et al., 2010; Guibinga et al., 2010; Yeh et al., 1998). Abnormalities of transcription and signaling factors controlling neural development have been documented for several additional cell models of HGprt deficiency including MN9D mouse neuroblastoma cells (Ceballos-Picot et al., 2009), mouse embryonic stem cells (Kang et al., 2013), human SH-SY5Y neuroblastoma (Kang et al., 2011), and human NT2 embryonic carcinoma lines (Guibinga et al., 2010). These results imply a developmental defect rather than a degenerative one.

Most of the cell models used in prior studies have low potential for delineating abnormalities responsible for neuronal development in the human LND brain. One study addressed this limitation by studying neural stem cells from aborted LND fetuses, which also suggested an abnormality of early development (Cristini et al., 2010). More recent studies have leveraged human induced pluripotent stem cells (iPSCs) for LND (Bell et al., 2021; Sutcliffe et al., 2021). These studies have shown that certain abnormalities can be detected at a very early stage in neural development, such as those relating to energy production. The purpose of the current studies was to further explore the value of an approach using neural stem cells (NSCs) derived from patient-based iPSCs for revealing potential developmental mechanisms that may be responsible for LND.

Methods

Cell cultures.

The current studies leveraged 6 recently described iPSC lines derived from 3 unrelated males with LND using a footprint-free mRNA transfection method (Sutcliffe et al., 2021). All procedures involving human subjects followed appropriate institutional and national guidelines and were approved by the Emory University Institutional Review Board. All studies were conducted in parallel with 6 iPSC lines derived from 3 normal healthy males. NSCs were prepared for all 12 lines. In brief, iPSCs were plated in a 6 well dish on Geltrex (GIBCO #A1413302) in mTeSR Plus medium on the first day. The following day, medium was changed to complete neural induction medium (GIBCO PSC Neural Induction Medium #A1647801). This medium was changed every other day until day 7, when NSCs were ready to be harvested and expanded. They were then plated on Geltrex-coated plates in neural expansion medium, which includes one part NeuroBasal Medium (GIBCO #21103049) to one part Advanced DMEM/F-12 (GIBCO #12634) with 2% neural induction serum (GIBCO #A1647701). Medium was changed every other day, and NSCs were further expanded or cryopreserved when they reached confluency. After at least 4 passages, they were stained for neural stem cell markers Nestin (Stem Cell Technologies #60091), SOX1 (R&D #AF3369), and SOX2 (R&D #MAB2018).

Gene expression.

Complete gene expression profiles were determined via RNA-seq as previously described (Sutcliffe et al., 2021). The procedure targeted 50 million paired-end, 100bp per library for 100 million total reads per sample. Across all samples, the percentage of uniquely mapped reads fell between 88 and 91%. After quality control checks and filtering genes with very low counts, a total of 15,822 genes were retained for quantitative analyses. Results from cultures for the LND and control NSC lines were interrogated for markers of neural stem cells, and any disease-related abnormalities.

For statistical comparisons, the Benjamini-Hochberg method for the False Discovery Rate (FDR) was used to correct for multiple comparisons. Results from NSCs were also compared to results from corresponding iPSCs previously published (Sutcliffe et al., 2021). Gene Ontology (GO) enrichment analyses of differentially expressed genes were done in ShinyGO v0.74 webserver (Ge et al., 2020). Genes with FDR<0.10 were considered for this analysis.

Protein expression.

Complete protein expression profiles were determined using shotgun proteomics as previously described (Sutcliffe et al., 2021). After quality control checks and filtering out peptides with low expression, a total of 24,865 peptides mapping to 2,280 proteins were retained for quantitative analyses. Results from cultures for the LND and control NSC lines were compared to delineate any disease-related abnormalities. Proteomics results for NSCs were also compared to results previously published for the corresponding iPSCs (Sutcliffe et al., 2021). For statistical comparisons the nominal uncorrected p-values were used, since the FDR method has been reported to be too conservative for exploratory proteomics (Pascovici et al., 2016).

Purine levels.

Purines were assessed using high performance liquid chromatography with photodiode array ultraviolet detection (HPLC-UV) and normalized to total protein using the Pierce BCA kit (Thermo Fisher Scientific, Rockford, IL) as previously described (Sutcliffe et al., 2021). The adenylate energy charge was calculated according to the formula: ([ATP] + 0.5 x [ADP])/([ATP] + [ADP] + [AMP]) which generates a parameter that varies between 0 and 1, with higher values indicating a higher energy charge (Atkinson, 1968; Shirley et al., 2007). Statistical analyses involved 2-way ANOVA with disease group as independent variable and each purine as dependent variable, with post-hoc Tukey test.

Results

Neural stem cells.

The LND (n=6) and control (n=6) NSCs had similar morphological appearances, with no obvious differences in overall growth rates. Immunostains for the pluripotency marker OCT4 were negative, but all lines showed positive immunostaining for markers typical of neural stem cells such as NES, SOX1, and SOX2. RNA-seq revealed the LND and control lines to express high levels of genes associated with neural stem cells, with no consistent differences between the LND and control lines (Figure 1A). As expected, pluripotency genes such as NANOG and POU5F1 were not expressed in the NSCs. These results indicate that HGprt deficiency does not prevent differentiation of NSCs.

Figure 1. NSC gene expression.

Figure 1.

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A) Genes typically expressed in neural stem cells. B) Volcano plot showing the spectrum of differences between LND and control lines, highlighting 6 genes significantly different at FDR < 0.05. C) Heatmap showing consistency of differentially expressed genes at FDR < 0.05. D) Gene Ontology (GO) functional enrichment analysis of 43 DEGs.

Differential gene expression in NSCs.

RNA-seq data were used to identify differences between the 6 LND and 6 control lines. The edgeR exact test revealed significant differences between LND and control lines for 43 transcripts (7 increased and 36 decreased) at FDR<0.10, and 6 transcripts (1 increased and 5 decreased) at FDR<0.05. The distributions of differences are shown as volcano plots (Figure 1B) and heat maps (Figure 1C). In these plots, HPRT1 was clearly reduced among the LND lines. Other significant changes included TMEM191B (transmembrane protein 191B with unknown function), PI4KAP1 (Phosphatidylinositol 4-Kinase Alpha Pseudogene 1), IFITM3 (Interferon Induced Transmembrane Protein 3), and ZP3 (Zona Pellucida Glycoprotein 3).

The gene expression data were then used to identify biological functions that differ between the LND and control lines. As might be expected for a housekeeping enzyme that plays a fundamental role in maintaining purine levels, numerous biological pathways were significantly affected when using Gene Set Enrichment Analyses with Gene-Ontology (GO) annotations. However, many of these different pathway findings were driven by the same group of WNT family genes (WNT3A, WNT4, WNT10B) along with PENK, CXCL14, and ZP3 (Figure 1D).

Because HGprt plays a role in purine metabolism, the data were also evaluated more specifically for changes in other enzymes of purine metabolism. Only 2 genes in the KEGG pathway for purine metabolism were significantly different in the LND vs control NSC lines at FDR<0.10. They included HPRT1 and PDE4C, which encodes a phosphodiesterase (not shown). These results are in line with prior studies that imply that most changes in purine metabolism that may be caused by HGprt deficiency are not regulated at the level of gene expression (Sutcliffe et al., 2021).

Protein expression in NSCs.

Proteomics methods were also used to identify differences between the 6 LND and 6 control NSC lines. The overall correlations among changes in protein and gene expression were in line with prior studies (Sutcliffe et al., 2021), and similar for the LND (R=0.50) and control (R=0.51) NSC lines.

A total of 118 proteins were significantly different between the LND and control NSCs (64 increased and 54 decreased) at p<0.05, and 22 proteins were significantly different (13 increased and 9 decreased) at p<0.01. With a cutoff of at least 1.5× change, and correction for multiple comparisons at FDR<0.10, no proteins reached statistically significant differences, not even HGprt, which is known to be absent in the LND lines. This finding confirms prior suggestions that the FDR method may be too conservative for discovery proteomics. The distribution of changes at p<0.01 are shown as Volcano plots (Figure 2A) and heat maps (Figure 2B). As expected, HGprt was absent from all LND lines, but readily detected in all controls.

Figure 2. NSC protein expression.

Figure 2.

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A) Correlation between the gene and protein expression results for both the LND and control lines. B) Volcano plot showing spectrum of differences between LND and control lines. C) Heatmap of showing consistency of 22 differentially expressed proteins at p < 0.01.

Comparison of results from NSC and iPSC.

Abnormalities in the expression of genes and proteins in the NSCs were next compared to findings previously reported for the corresponding iPCS lines from the same individuals (Sutcliffe et al., 2021). Despite the large numbers of differentially expressed genes when comparing LND and control lines, only 9 were shared across both cell types (Figure 3A). As expected, HPRT1 was among the reduced transcripts for both NSCs and iPSCs, a finding confirmed by qPCR. The limited overlap between NSCs and iPSCs confirm that the consequences of HGprt deficiency vary according to state of differentiation. Because 2 independent iPSC lines were derived from each of 3 individual LND cases or controls, the impact of the relationships between biological replicates from the same case was further evaluated using principal component analyses. At the iPSC stage, these plots revealed substantial overlap among the lines, with distinct but limited clustering of some pairs from the same case (Figure 3B). In addition, this pairwise clustering varied according to differentiation state. For example, both iPSC lines from case L1 tended to cluster together apart from the other lines. For the NSCs, both lines from C3 clustered separately from the remaining lines. Because of the substantial overlap among all the lines, results from pairs of clones from the same individual were not merged together as if they were a single biological replicate.

Figure 3. Comparison of NSCs and iPSCs.

Figure 3.

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A) Significant differences between LND and control genes in iPSCs or NSCs. B) Principal component analyses for RNA-seq results from iPSCs and NSCs. C) Significant differences between LND and control proteins in iPSC or NSCs. Only 9 genes and 9 proteins overlapped these two cell states. D) Boxplots showing protein changes between control and LND NSCs for subunits of ATP synthase in both iPSCs and NSCs. Those significant at p<0.01 are shown in bold, while those not significant are shown in regular font.

For the proteomics, 9 differentially expressed proteins were shared across both cell types (Figure 3C), again confirming that consequences of HGprt deficiency may vary according to state of differentiation. As expected, a decrement in HGprt protein was one of the findings shared across NSCs and iPSCs, a finding confirmed by Western blot. Another consistent abnormality involved 3 distinct subunits of the mitochondrial F1F0 ATP-synthase (Figure 3D), which were always significantly higher in the LND iPSCs and NSCs compared to their respective controls. The proteomics data were therefore evaluated in more detail for all F1F0 ATP-synthase subunits. For both iPSCs and NSCs, the median value for the LND lines was always higher than the control lines, even when the difference did not reach statistical significance for some of the subunits. Since these results imply a higher energy demand for the LND lines, ATP levels were measured along with other purines so that it was possible to calculate the energy charge of the cells. For both iPSCs and NSCs, there were only small changes between LND and control lines for most purines, including ATP (Figure 4A,D). The energy charge showed minimal change between the LND and control lines (Figure 4 B,E). However, extracellular hypoxanthine was markedly elevated in LND lines compared to controls for both iPSC and NSCs (Figure 4C,F), confirming failure to recycle main HGprt substrate in the LND lines.

Figure 4. Purines and energy.

Figure 4.

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A) Changes in intracellular purine levels in LND iPSCs. Results are expressed as percent of matched controls (average ± SEM, n=6 per group), and only those purines measured at reliably detectable levels are shown. B) Changes in extracellular purine levels in LND iPSCs. C) Adenylate energy charge (AEC) and guanylate energy charge (GEC) in LND iPSCs. D) Changes in intracellular purine levels in LND NSCs. E) Changes in extra-cellar purines in LND NSCs. F) Adenylate energy charge (AEC) and guanylate energy charge (GEC) in LND NSCs. Asterixes show significant differences for the LND group for extracellular hypoxanthine only, in both iPSCs and NSCs.

Discussion

These studies delineate consequences relating to the loss of HGprt-mediated purine salvage for gene and protein expression during early development of neural stem cells from human subjects with LND. They show several important new findings. First, NSCs from LND can be readily generated with characteristics and growth habits similar to control NSCs, indicating that the loss of HGprt does not prevent early specification or viability of NSCs. Second, the LND NSCs show multiple consistent abnormalities of gene and protein expression, indicating that the loss of HGprt has a significant impact on many functions of immature neurons. Third, abnormalities in gene and protein expression are quite distinct in LND NSCs versus iPSCs, indicating that the consequences of HGprt deficient depend on the state of differentiation.

The gene expression studies revealed that HGprt deficiency significantly affects multiple transcripts with diverse functions in NSCs. One consistent biological pathway impacted in LND NSCs related to WNT signaling (Figure 1D). The WNT family encodes a group of related secreted glycoproteins that are known to play an important role in early neuronal development. Interestingly, several prior studies have implicated the WNT signaling in LND. Kang and colleagues described abnormal WNT/β–catenin signaling in human SH-SY5Y neuroblastoma cells rendered HGprt deficient via transfection with a short hairpin RNA targeting HPRT1 (Kang et al., 2013). Torres and Puig described abnormal WNT/β–catenin signaling in NTERA-2 embryonic carcinoma cells exposed to high hypoxanthine levels often seen in LND patients (Torres & Puig, 2015). Jiang and colleagues showed that over-expression of HPRT1 in the mouse brain altered WNT/β–catenin signaling (Jiang et al., 2020). All together, these studies provide converging evidence for dysregulation of WNT signaling in LND. These observations are relevant, because WNT signaling is known to play a key role in the development and function of dopamine neurons (Oliva et al., 2018; Wang et al., 2020), which are believed to be among the most vulnerable neurons in LND (Egami et al., 2007; Goettle et al., 2014; Jinnah et al., 1999; Jinnah et al., 1994; Visser et al., 2000). The mechanism by which HGprt deficiency might affect WNT pathways remains to be determined. This mechanism is not likely to reflect a direct effect of the HPRT1 mutations on the expression of these genes, but an indirect effect of the consequences of HGprt deficiency for purine metabolism. In any event, the replication of abnormalities in WNT pathways in these new LND NSCs provides valuable confirmation of prior findings from other types of cells, and a valuable new tool for exploring potential mechanisms.

The protein expression studies similarly revealed HGprt deficiency to affect multiple proteins with diverse functions. For both NSCs and iPSCs, one striking finding was a consistent increase in several subunits of the mitochondrial F1F0 ATPase. Also known as Complex V, the F1F0 ATPase is a multi-subunit protein that plays a key role in ATP synthesis in mitochondria. These findings are consistent with recent studies that have implied HGprt deficiency may impact cellular energy demands (Bell et al., 2021). Similar to the gene expression studies above, the changes in protein expression are not likely to be a direct effect of the HPRT1 mutations. Instead, they are more likely compensatory responses to the biochemical consequences of HGprt deficiency. ATP is quantitatively the most abundant purine in most cells (Traut, 1994), so one potential explanation is that the failure of HGprt-mediated purine recycling leads to ATP deficiency. However, measurement of ATP showed no significant decrement in the LND lines (Figure 4A,D). Another potential explanation is that failure of HGprt-mediated purine recycling leads to purine wasting, and replenishment of purines via the de novo synthetic pathway is energetically costly, so LND lines have a higher energy burden. Measures of extracellular purines confirmed marked wasting of hypoxanthine (Figure 4C,F), while measures of energy state imply the LND lines have compensated for this loss (Figure 4B,E). Although a review of many prior studies revealed no consistent decrement in purine nucleotides among HGprt-deficient cells (Shirley et al., 2007), one recent study suggested that purine nucleotide deficiency or energy impairments may emerge later in neuronal development (Bell et al., 2021). These findings confirm that HGprt deficiency may have different consequences among different types of cells, including different states of differentiation. Therefore, understanding how HGprt deficiency leads to the neurodevelopmental abnormalities may require more detailed studies of its impact more specifically on developing neurons. The current studies suggest that a strategy involving human iPSCs could provide a tool to do this.

Acknowledgements

The authors declare no conflicts of interest related to this work. This work was supported by the Lesch-Nyhan Syndrome Children’s Research Foundation, the National Institute for Neurological Diseases and Stroke (NINDS) at the National Institutes of Health (NIH) (R56 NS102980, R01 NS109242, R01 NS119758), the Dutch Research Council (NWO ZonMW/Veni grant 916.12.167), the Dutch Brain Foundation (Hersenstichting Fellowship (F2014(1)-16) and the LND Famiglie Italiane Onlus. Additional support came from the Emory Integrated Genomics Core and the Emory Integrated Proteomics Core, both of which are subsidized by the Emory University School of Medicine.

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