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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Huntingtin hPSC (Unraveling huntingtin function in cortical and striatal human development)

Teaser

Summary

Huntington\'s disease (HD) is a rare neurodegenerative autosomic dominant disorder which main symptoms include severe motor dysfunction and cognitive deficits. Genetically, HD is a monogenic disorder driven by an expanded CAG repeat mutation located at the 5\' end of the huntingtin gene (Htt) that results in increased dysfunction and cell death of striatal medium spiny neurons (SMN) and certain cortical areas. Although HD main symptoms are manifested at mid-life (around 35 years of age), it has been suggested that early neurodevelopmental defects may be present in the patients that could contribute to the late pathology.
The huntingtin gene is ubiquitously expressed in all cells and tissues and it has been shown that its complete loss-of-function leads to early embryonic death due to defects in extraembryonic tissue organization. To circumvent this early lethal phenotype and to focus on the brain phenotype, several conditional knock out and heterozygous mouse models have been consequently generated. The specific cellular and molecular role of huntingtin at different stages of cortical development has also been addressed by conditional knock out mouse models that specifically removed huntingtin from the brain, for instance at an early stage, under an early progenitor promoter. However, to date, no studies have addressed and dissected the different roles of the human huntingtin protein in human cortical and striatal development specifically in the progenitor and neuronal populations.

The main goal of this project is the generation of new cellular models to study huntingtin (HTT) function during human brain development in specific neuronal subpopulations using reductionist approaches in a purely in vitro setup through the generation of several Htt conditional and full knock out pluripotent stem cell (PSC) lines.
The completion of this project would add to the existing field of research new cellular tools to study huntingtin function in human cells in a time and cellular type-specific controlled fashion. This project will shed some light into a long overlooked topic in HD field, which is the developmental role of huntingtin in the brain, specifically in the cortex and striatum, the two areas that are mostly affected in HD patients.
Through this work we will test if developmental defects are caused by the specific loss-of-function of Htt in different neuronal populations in early and late stages in 2D and 3D neuronal cultures.
The specific objectives of the present project are:
1) The generation of huntingtin full knock out and conditional knock out pluripotent stem cell (PSC) lines
2) Screening of the phenotypes caused by huntingtin loss-of-function
3) Analysis of the transcriptomic changes caused by loss-of-function of huntingtin

Work performed

We have generated several heterozygous full knock out (HTT -/+) H9 hPSC lines through CRISPR-CAS9 mediated technology and the use of two synthetic gRNA guides to target huntingtin locus. These huntingtin cellular tools showed decreased levels of huntingtin during stem cell renewal conditions, as expected. Importantly, these experiments also suggest that huntingtin function might be essential during stem cell renewal in human pluripotent stem cells as biallelic targeting of the HTT locus to generate HTT -/- PSC lines was not feasible.
We also report the generation of the heterozygous huntingtin conditional knock out hPSC lines using novel technology for one-step reversible conditional gene knock-outs. This technology follows a knock-in strategy using CRISPR-CAS9 and single or double guide gRNAs combined with an invertible cassette (FLIP-FlpE) flanked by loxP sites. The FLIP-FlpE cassette in the original orientation is â€œsilencedâ€ and spliced out and therefore does not interfere with normal huntingtin expression. However, upon CRE recombination the cassette is inverted and excised towards a â€œmutagenic orientationâ€ resulting in huntingtin depletion from the cells. These experiments resulted in the generation of several HTT cko/+ lines that showed in the original â€œnon-mutagenic orientationâ€ not to affect HTT normal levels. Upon CRE nucleofection these lines correctly showed decreased huntingtin levels of expression, as expected. Interestingly, we also detected an important reduction in the expression of several cell renewal genes.
These results suggest that huntingtin function might be involved in the maintenance of stem cell renewal in human pluripotent stem cells, and that at least some of the stem cell renewal genes expression are affected upon HTT lowering strategies.

Overall, we have generated several human cellular embryonic stem cell models that will be instrumental for the future study of huntingtin function in vitro. These data could be instrumental for the future study of HTT loss-of-function in neuronal subpopulations in the cortex and striatum during development, and it could be very relevant for the study of the potential side effects of total HTT lowering strategies in adult neurons during current HD clinical trials.

Final results

Numerous biological and cellular roles have been described for huntingtin protein, however to date it is unclear which of these roles are physiological and which are essential for different cell types in the brain. Moreover, huntingtin function has mostly been studied during adulthood; however, huntingtin expression is essential for the early embryo as complete huntingtin loss-of-function leads to early lethal phenotype. Therefore the identification of phenotypes caused by the loss of Htt during development is of extremely importance. The present project contributes to the field of Huntingtonâ€™s disease in unraveling and dissecting the role/s of huntingtin in cortical and striatal human brain development and neuronal function using embryonic stem cells as an in vitro tool.
The present project developed numerous cellular tools using human embryonic stem cells and CRISPR/Cas9 mediated technologies that will be very useful for the continuation of this project and for future possible studies in the field. This project also used a recently described technology for the generation of one-step reversible conditional knock out genes in human cells, which is of great importance for the human embryonic stem cell and gene editing fields. The application of this technology and these developed cellular tools will allow the study of the effects of a gene loss-of-function in a controleed time manner and in specific human neuronal subpopulations, much like what has been done previously in rodent cells through the use of genetic mouse models.

Moreover, current clinical trials for Huntingtonâ€™s disease patients rely mostly on the downregulation of total (wild-type and mutated) huntingtin levels through the use of gene-silencing agents. The leading HTT-lowering technology is currently in phase III of clinical trials and relies on an antisense approach that targets all forms of HTT messenger RNA. However, HTT protein is highly conserved between different species and has been proven to show essential roles in neuronal survival (BDNF expression), mitochondrial function, gene transcriptional regulation, cellular transport, etcâ€¦ Therefore, this project holds also potential for future societal implications, as the study of the effects of huntingtin loss-of-function in different neuronal subpopulations could be instrumental for the understanding of putative side effects from ongoing HD clinical trials.