How CTIP2 deficiency drives medium spiny neuron degeneration and dysfunction: implications in Huntington's disease pathogenesis

Huntington's disease (HD) is an increasingly prevalent and progressively disabling type of dementia in our ageing society. This neurodegenerative disorder is caused by a cytosine-adenine-guanine (CAG) trinucleotide expansion in the Huntingtin gene. Apart from being a movement disorder, HD is also characterised by severe dementia including impairment of mood and thinking skills and a loss of memory severe enough to interfere with daily life. The impact of HD on patients and their families as well as its burden on health care systems is growing exponentially. Currently there is no cure or disease modifying treatment to delay the onset and progression of this disorder. Striatal degeneration, specifically loss of medium spiny neurons (MSNs), is hallmark pathology of HD. Despite advances in disease modelling using genetically modified animals and patient-derived human induced pluripotent stem cells (hiPSCs), little is known about the precise mechanism underlying this selective HD pathology which impedes design of appropriate and effective therapies.
Recent studies, including pilot data of our own, suggest that a striatal transcription factor COUP TF1-interacting protein 2 (CTIP2) plays an important role in the specificity of neurodegeneration in HD. In light of these findings, we propose to further demonstrate that CTIP2 deficiency induces human MSN pathogenesis, and to establish the extent of this mechanism contribute to HD pathogenesis. Understanding the control of MSN neuron functional maintenance is of paramount importance in order to characterise and reverse disease phenotypes.
In this project, we will make striatal neurons from human embryonic stem cells (hESCs) engineered to carry a dysfunctional form of CTIP2 and healthy control hESCs expressing normal CTIP2. We will investigate in detail the cellular pathology caused by CTIP2 dysfunction and workout the underlying molecular mechanisms. Moreover, we will establish that striatal neuronal deficits caused by CTIP2 contribute to HD pathogenesis. It is believed that these findings will help to identify novel targets for therapeutic interventions that may provide means to modify HD progression.

Dysfunction and neurodegeneration of striatal GABAergic medium spiny neurons (MSNs) is the core pathophysiological hallmark of Huntington's disease (HD). Increasing lines of evidence implicates impairments of MSN specification and maturation during development as major contributors to HD. Also, reduced CTIP2 transcription factor level and together with its dysfunction in the striatum are thought to contribute to HD pathogenesis. However, due to our limited knowledge of human striatal development and a lack of defined aetiology and causal mechanisms of pathogenesis, there is currently no effective treatment for this disorder.
We have previously developed a reliable method for inducing MSNs from human embryonic stem cells (hESCs) and also generated CTIP2 mutant ESC lines using CRISPR technology. We found that CTIP2KO MSNs exhibited HD-like cellular pathologies compared to MSNs derived from isogenic control hESCs. Moreover, genome-wide RNAseq analysis of CTIP2KO MSNs revealed a significant overlap between CTIP2 regulated genes and those altered in HD iPSC-derived neurons and mouse models of HD, providing direct evidence that CTIP2-deficicy leads to MSN pathogenesis.
Therefore, we will in this proposal determine how CTIP2 hypofunction leads to neuronal death and MSN dysfunction, and the extent of which contribute to HD pathogenesis, using a combination of CTIP2KO hESCs, HD iPSCs and animal models of HD. This work will determine gene regulatory networks and signalling pathways in striatal neurons governed by CTIP2 and provide insight into mechanisms behind cellular abnormalities in HD MSNs. The knowledge obtained from these studies will facilitate the development of new and more effective treatments for HD.

The project is anticipated to impact on several intellectual and scientific fronts in stem cell neurobiology and disease modelling (see Academic Beneficiaries). A significant contribution will be the new knowledge on the molecular control of striatal medium spiny neuron fate specification and maintenance. Understanding how cells acquire specific neuronal cell fate is of great importance to the growing field of stem cell research and translational medicine. This is of particular relevance to Huntington's disease where aberrant striatal development and subsequent neurodegeneration have been strongly implicated.

The impact of neurodegenerative and psychiatric disorders on patients and their families as well as its burden on health care systems is growing exponentially. Currently there is no cure or disease-modifying treatment to delay the onset and progression of Huntington's disease (HD). However, the development of new treatments has been limited both by our imperfect understanding of the development and function of striatal neurons and the lack of a defined aetiology and limited pathophysiology of HD. Therefore, there is a societal urgency for a better understanding of the molecular basis causing HD that will ultimately allow for a more rational design of new therapeutic alternatives to target this increasingly prevalent and disabling type of dementia in our ageing society. This project will help us to understand governance of MSN gene regulatory network by a transcription factor CTIP2 and how perturbations in CTIP2 function contribute to HD pathophysiology. Establishment of the exact mechanisms underlying CTIP2 deficiency-induced MSN pathogenesis in human neurons and the extent of overlap between these cellular abnormalities and HD phenotypes will directly impact on developing strategies for effective therapeutic interventions. Approaches targeting the CTIP2 pathway thus may ameliorate cellular and neurochemical changes in the striatum and cerebral cortex responsible for neuronal dysfunction in HD.

We anticipate that the project will also provide a valuable foundation for exploring hESC-derived neuronal network formation in "a dish" in modelling human diseases. Since HD preferentially affects MSNs, an experimental model of the disorder implies achieving cultures of functional MSNs able to establish synaptic connections between themselves and with biologically relevant afferent and efferent neurons. Neuronal differentiation of hESCs and establishment of MSN-cortical and MSN-dopamine neuronal co-cultures offer a unique opportunity to model these synaptic connections in vitro and investigate how MSN dysfunction affects these processes.

The themes of this proposal - stem cell biology and disease modelling - are medically and economically important fields for the UK and are expected to grow exponentially as genomic data and stem cells are exploited and therapeutic interventions begin to rely on this knowledge. Thus there is a great need for trained researchers with experience in appropriate fields of research. Specialised stem cell research is developing at very high rate and requires highly trained personnel to support this. Personnel with skills and knowledge of stem cell, developmental and molecular biology are likely to play a key role in the post-genomics era. This project will contribute to the training of such personnel and will allow the dissemination of this knowledge and skills.