Decoding the molecular identity of neural stem cell types

The development and function of the brain depend on the continuous production of new brain cells (neurons and glia) from progenitors called neural stem cells. This process is not only important to form the brain but is also crucial throughout our adult lives, as tasks such as learning and memory rely on the performance of newly adult-born neurons. We are witnessing an increasing incidence of long-term brain disorders, ranging from neurodegeneration (e.g. Alzheimer's disease), psychiatric (e.g. major depressive disorder) to neuro-oncological (brain tumours) conditions. Due to the increase of these life devastating and costly illnesses, the development of novel and more effective therapies is imperative, as the existing treatments only ameliorate but do not eradicate the majority of conditions. Neural stem cell-based therapies offer new hope for such patients, with the goal of supplying well-functioning brain cells, eliminating mal-functioning cells and/or replacing lost cells. However, one major impediment for the development of these therapies is that we currently know little of the basic properties of neural stem cells in vivo, that is, how they are activated and controlled in the living brain. Aggravating these problems is the fact that different kinds of neural stem cells co-exist in the brain, and behave in different ways. Currently, we are unable to understand these diverse brain neural stem cell types, how they develop and are regulated, as there are not enough specific markers available allowing their identification.

In this project, we propose to identify and characterise the function of novel neural stem cell markers by taking advantage of one of the best genetic model organisms available to scientists, the fruit fly Drosophila. More than 75% of human disease genes have a match in Drosophila, and the principles ruling neural stem cell and brain development have been proven to be remarkably similar. Thus, the Drosophila model is a powerful tool to uncover the properties of neural stem cell types, and unlike other laboratory animal models such as mice, we can easily identify, manipulate and study neural stem cells in live fruit fly brains.
We will harvest from the living Drosophila brain different neural stem cell types and compare the transcripts they express, that is, their genetic instructions' content. In this way, we will obtain a list of transcripts specific for different kinds of neural stem cells. We will next characterise the function of selected transcripts by examining their role in the behaviour of the distinct neural stem cell types, such as their ability to produce new cells. Finally, we will examine if mammalian transcripts analogous to the ones identified in Drosophila also mark distinct neural stem cell populations in the mouse brain, a system even more similar to the human brain.

Our team including two national collaborators from Plymouth and Cambridge Universities, and an international project partner from the Scripps Research Institute in the USA, has in place the required expertise and materials to perform successfully and timely the proposed work. In sum, given the high similarity between the Drosophila and mammalian nervous systems, we anticipate our work to significantly contribute to clarify number and molecular identity of neural stem cells types, a central question in stem cell biology with clear medical implications, and of relevance to seed the development of future neural stem cell therapies.

Brain function relies on the continuous generation of neurons and glia from Neural Stem Cells (NSCs). It is becoming clear that the developing and adult brain harbours NSC types with distinct self-renewal, differentiation potential and temporal regulatory control. In vivo clonal analysis in mammalian models has been crucial to the study of NSC traits, but this retrospective analysis implies prior knowledge of the transcripts distinct NSCs express in order to trace them. Given the presence of multiple precursor types in the mammalian brain and our poor understanding of their molecular identity, it is advantageous to make additional use of an evolutionary conserved model system, in which the location of different NSC types and lineages are known, and their development can be easily followed in vivo via permanent lineage tracing such as the fruit fly Drosophila central nervous system.
We propose to identify transcripts conferring molecular identity to two distinct NSC types in the Drosophila larval brain at two developmental time windows. We will employ our successfully established single-cell transcriptome microarray analysis technique (Bossing et al., 2012) to harvest and compare the transcriptome of single type I and type II NSCs removed from live brains. This high precision single cell approach avoids chasing after false candidates due to mixed cell and genetic backgrounds, non-physiological cell cultures or sorting procedures. After expression validation of candidate transcripts, functional analysis of selected candidates will be performed using gain- and loss-of-function genetics. We will also establish the expression profile of identified selected mammalian orthologues in neurogenic regions of the mouse brain. Our work is expected to significantly contribute towards resolving the current molecular identity quest of NSC types, a central question in stem cell biology with medical implications.

This work will directly benefit the UK and international academic community and the wider public with an interest in ours and associated broader research areas. Its impact has the potential to also seed future research lines in more applied biomedical research such as in stem cell therapy and biomarker development.

Impact on the Academic community: We will publishing our results in open access, peer-reviewed journals, communicate and discuss our work in seminars, national and international conferences, and participate in research information events open to all academics. In this way, we will maximize outreach, encourage potential collaborations and make our work available to academics worldwide. The technical aspects, nature and wealth of the data to be produced may be of interest and benefit diverse disciplines, such as genome biology, developmental biology, neurobiology, stem cell biology and cancer biology. It may also potentially contribute to the seeding of future research lines on more applied biomedical research areas such as in stem cell therapy and biomarker development.
Of note, our microarray datasets will in addition be freely accessible on ArrayExpress and via a Java applet on the PI laboratory web site, allowing users to identify transcripts of which the expression changes between different neural stem cell types, and/or within the same neural stem cell type over time. In addition, it will reveal the transcripts' mouse and human orthologues, as well as its potential functions. We believe that such a database will become a basic tool for researchers studying neural stem cells, benefitting additionally broader related areas as mentioned above.

Impact on general Public: We recognise that increased knowledge about neural stem cells is of general public interest due particularly to the economical and medical potential of neural stem cell-associated therapies. We will convey our work, and the significance of a better understanding of neural stem cell properties, to the wider public in outreach and engagement activities, ensuring the information is communicated in an accurate but accessible lay language.
While the proposed work is directed at basic laboratory research, it has the potential to seed in the future more applied biomedical research areas, such as in stem cell therapy and biomarker development. We will therefore assess the results for potential IP, and approach our Research, Enterprise and Innovation Office if appropriate.

Employability: The PI will initially undertake most of the outreach/public engagement activities but will encourage and progressively increase the involvement of the PDRA. The PDRA will in addition obtain valuable transferable skills such as data management, usage of equipment, working towards deadlines, team-working, oral and written presentations, purchasing and budgeting. Together, these skills will make him/her an asset to all employment sectors. He/she will also be encouraged to participate in training courses for career development free of charge and regularly available by two independent Plymouth University services: The Research Support Program and The Research Development Program. Finally, the PDRA will be from start integrated in an working team with national and international project partner/collaborators renowned in their fields, will also have the chance to interact with other academics in seminars and conferences, and be encouraged to build his/her own research network.