Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing alterations in the lysosomal pathway

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Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an excessive expansion of a CAG trinucleotide repeat in the gene encoding the protein huntingtin, resulting in an elongated stretch of glutamines near the N-terminus of the protein. Here we report the derivation of a collection of 11 induced pluripotent stem (iPS) cell lines generated through somatic reprogramming of fibroblasts obtained from the R6/2 transgenic HD mouse line. We show that CAG expansion has no effect on reprogramming efficiency, cell proliferation rate, brain-derived neurotrophic factor level, or neurogenic potential. However, genes involved in the cholesterol biosynthesis pathway, which is altered in HD, are also affected in HD-iPS cell lines. Furthermore, we found a lysosomal gene upregulation and an increase in lysosome number in HD-iPS cell lines. These observations suggest that iPS cells from HD mice replicate some but not all of the molecular phenotypes typically observed in the disease; additionally, they do not manifest increased cell death propensity either under self-renewal or differentiated conditions. More studies will be necessary to transform a revolutionary technology into a powerful platform for drug screening approaches.

Highlights

► We model Huntington's disease (HD) through induced pluripotent stem (iPS) cells. ► We generated a large number of iPS cell lines from a HD genetic mouse model. ► HD-iPS cells showed similar behavior in somatic reprogramming and cell cycle rate. ► Alterations in cholesterogenic genes and lysosomal biogenesis were found. ► Neurons differentiated from HD-iPS cells contained huntingtin aggregates.

Introduction

Huntington's disease (HD) is an untreatable, progressive genetic neurodegenerative disorder caused by an unstable expansion of the CAG repeat within the coding region of the IT-15 gene. This gene encodes for the huntingtin (HTT) protein and the mutation results in an elongated stretch of glutamines in the N-terminus of the protein (HD Collaborative Research Group, 1993). This HTT mutation is responsible for massive brain neurodegeneration characterized by the prevalent loss of efferent medium spiny neurons in the striatum, the main input station of the basal ganglia circuit, but progressively involves cortical neuronal structures as well (Reiner et al., 1988, Rosas et al., 2003, Rosas et al., 2005, Rosas et al., 2008).

HD is characterized by chorea, cognitive abnormalities, and psychiatric disturbances that manifest in mid-adulthood and progress inexorably toward death. The CAG expansion in HTT is the triggering event that endows the protein with new toxic functions that are deleterious to striatal and cortical neurons. At the same time, the specificity of the neuronal loss may be due to the protein context in which the CAG is expanded (Cattaneo, 2001); the mutation also affects the ability of normal HTT to exert beneficial activities for the neurons that degenerate in HD (Zuccato et al., 2010).

While our understanding of HD pathophysiology is advancing rapidly, our knowledge is still incomplete. Since HD is caused by a single mutation, introduction of the mutant gene into non-human primates, mice, flies, fishes, and worms has generated disease models that have been extremely valuable in the identification of pathways affected by the mutation and as validation tools for ex vivo-identified targets (Zuccato et al., 2010). In parallel, since the year 2000 we and others have reported the generation of a large collection of brain-derived cell lines from rodents, including the first clones of immortalized ST14A cells carrying the full length or truncated normal or mutant HTT gene (Rigamonti et al., 2000), their inducible variants (Sipione et al., 2002), knock-in cell lines (Trettel et al., 2000), and, more recently, rodent- and human-derived neural stem cell lines carrying the mutant HTT gene (unpublished results).

Collectively, these and other cell lines have been instrumental in providing the first indications of a loss-of-HTT-function mechanism operating in HD, and were used to deliver the first evidence of losses in BDNF mRNA and protein levels and reduced transcription of other NRSE-RE1-controlled neuronal genes (Rigamonti et al., 2007, Zuccato et al., 2003, Zuccato et al., 2007). In addition, gene expression profiling of ST14A cell line stably expressing an inducible mutant HTT construct revealed a defect in the cholesterol biosynthesis pathway that was confirmed in multiple mouse models; that study is currently the focus of both investigations and debate (Valenza and Cattaneo, 2011, Valenza et al., 2010).

Importantly, somatic reprogramming technology (Blelloch et al., 2007, Takahashi and Yamanaka, 2006, Takahashi et al., 2007) has yielded iPS cells with the potential to bridge clinical knowledge in patients with the molecular and biological expertise in genetically precise human cell lines that, upon differentiation, should more closely resemble the neurons affected by neurodegenerative disease. The generation of patient-specific cell models offers a number of advantages for neurological diseases research (Abeliovich and Doege, 2009, Brennand et al., 2011, Dimos et al., 2008, Ebert et al., 2009, Park et al., 2008, Soldner et al., 2009), although this technology may be limited by the difficulty of recapitulating disease progression in a dish.

Here we report the generation of a collection of 40 mouse iPS cell clones derived from two fibroblast lines obtained from R6/2 mice (HD-iPS cell lines) and 20 iPS cell lines from one wild type fibroblast line (the wt-iPS cell line). R6/2 mice were generated by insertion of a 1.9-kb DNA fragment containing the human HTT promoter and exon 1 of the human HTT gene bearing 144 CAG repeats (Mangiarini et al., 1996). These mice exhibit severe behavioral and anatomical symptoms, including widespread nuclear inclusions of mutant HTT in neurons (Davies et al., 1997, Morton, 2000) and typical gene expression dysregulation (Apostol et al., 2008, Conforti et al., 2008, Luthi-Carter et al., 2000, Luthi-Carter et al., 2002, Sadri-Vakili et al., 2007, Sipione et al., 2002, Tarditi et al., 2006, Valenza et al., 2007, Zuccato et al., 2001, Zuccato et al., 2005, Zuccato et al., 2010). We analyzed these wt- and HD-iPS cells and found that while somatic reprogramming is not affected by the HD mutation, typical HD cellular features are present in HD-iPS cells but not in the wt-iPS lines, mainly involving transcriptional dysregulation in cholesterol biosynthetic genes and lysosomal pathway activation.

Section snippets

iPS cell generation

The day before transfection, PLAT-E cells (Morita et al., 2000) were seeded at a density of 4 × 106 cells/100-mm dish. The next day, pMXs-based retroviral vectors (Klf4, Sox2, c-Myc, and Oct4, Addgene plasmid 13370, 13367, 13375, 13366, respectively) were introduced into PLAT-E cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Six hours after transfection, the medium was replaced with PLAT-E medium without selection.

TTFs were seeded at 1.2 × 105

Generation and pluripotency evaluation of mouse HD-iPS cells

TTFs were obtained from 10-week-old R6/2 mice and transduced with four reprogramming factors (Klf4, Sox2, c-Myc, and Oct4) in a single retroviral infection. After three weeks under mouse ES cell-supporting conditions, compact mouse ES-like colonies emerged from a background of fibroblasts, as previously described (Blelloch et al., 2007, Nakagawa et al., 2008, Takahashi and Yamanaka, 2006). HD-iPS cells were generated from two HD fibroblasts cell lines, HD2 and HD4, obtained from two different

Discussion

The advent of iPS cell technology (Takahashi and Yamanaka, 2006) has opened the door to generating patient-specific sources of donor cells for transplantation approaches that may preclude immunorejection, although a cautionary note to this aspect was recently raised (Zhao et al., 2011). More realistic hopes lie in the unprecedented opportunity to recapitulate pathological tissue formation in vitro, thereby enabling the investigation of complex questions related to neurobiology, disease-specific

Conclusions

iPS cells can be generated through somatic reprogramming of readily accessible tissue from patients with any condition. The obvious advantage of such an approach is that patient-specific iPS cells carry the precise genetic variants, both known and unknown, involved in the disease. Additionally, patient-specific iPS cells may eventually serve as a customizable resource for personalized regenerative medicine, drug testing, and predictive toxicology studies. Here we have reported a

Acknowledgments

This research was supported by initial funding from Fondazione Cariplo (Italy) in the context of the Operational Network for Biomedicine par Excellence in Lombardy project entitled “A genetic toolkit for the analyses of neural stem cells—acronym: Mouse NS-toolkit.” This work was also supported by Ministero dell'Istruzione dell'Universita` e della Ricerca (MIUR, 2008JKSHKN to E. Cattaneo) and partially by NeuroStemcell (European Community's Seventh Framework Programme grant agreement nr. 222943

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