Mono- and Biallelic Inactivation of Huntingtin Gene in Patient-Specific Induced Pluripotent Stem Cells Reveal HTT Roles in Striatal Development and Neuronal Functions.

Background: Mutations in the Huntingtin (HTT) gene cause Huntington's disease (HD), a neurodegenerative disorder. As a scaffold protein, HTT is involved in numerous cellular functions, but its normal and pathogenic functions during human forebrain development are poorly understood. Objective: To investigate the developmental component of HD, with a specific emphasis on understanding the functions of wild-type and mutant HTT alleles during forebrain neuron development in individuals carrying HD mutations. Methods: We used CRISPR/Cas9 gene-editing technology to disrupt the ATG region of the HTT gene via non-homologous end joining to produce mono- or biallelic HTT knock-out human induced pluripotent stem cell (iPSC) clones. Results: We showed that the loss of wild-type, mutant, or both HTT isoforms does not affect the pluripotency of iPSCs or their transition into neural cells. However, we observed that HTT loss causes division impairments in forebrain neuro-epithelial cells and alters maturation of striatal projection neurons (SPNs) particularly in the acquisition of DARPP32 expression, a key functional marker of SPNs. Finally, young post-mitotic neurons derived from HTT-/- human iPSCs display cellular dysfunctions observed in adult HD neurons. Conclusions: We described a novel collection of isogenic clones with mono- and biallelic HTT inactivation that complement existing HD-hiPSC isogenic series to explore HTT functions and test therapeutic strategies in particular HTT-lowering drugs. Characterizing neural and neuronal derivatives from human iPSCs of this collection, we show evidence that HTT loss or mutation has impacts on neuro-epithelial and striatal neurons maturation, and on basal DNA damage and BDNF axonal transport in post-mitotic neurons.

Modeling and Targeting Neuroglial Interactions with Human Pluripotent Stem Cell Models.

Generation of relevant and robust models for neurological disorders is of main importance for both target identification and drug discovery. The non-cell autonomous effects of glial cells on neurons have been described in a broad range of neurodegenerative and neurodevelopmental disorders, pointing to neuroglial interactions as novel alternative targets for therapeutics development. Interestingly, the recent breakthrough discovery of human induced pluripotent stem cells (hiPSCs) has opened a new road for studying neurological and neurodevelopmental disorders "in a dish". Here, we provide an overview of the generation and modeling of both neuronal and glial cells from human iPSCs and a brief synthesis of recent work investigating neuroglial interactions using hiPSCs in a pathophysiological context.

Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay.

The neurodegenerative Huntington's disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.