Harnessing natural cellular variability to understand how neurons maintain their axodendritic polarity

The different parts of your body all play different roles in keeping you alive and healthy. But dividing jobs between different spatial compartments like this doesn't just happen at the level of the whole organism - many of your individual cells do this too. This phenomenon of compartmentalising functions within distinct sub-cellular zones is known as 'polarity', and it reaches its peak in your brain cells, or neurons. Most neurons are broadly divided into two major polar specialisations: dendrites and axons. These have distinct structural and functional attributes that are crucial for information flow within neuronal circuits. Understanding how individual neurons organise themselves into axons and dendrites is therefore vital for understanding how a healthy brain operates. However, while we know a good amount about the processes involved in setting up neuronal polarity in the first place, we currently know a lot less about how neurons keep their polarity intact afterwards. How do neurons make sure that their axons stay axons, and their dendrites stay dendrites? Given that your brain cells are normally about as old as you are, maintaining polarity like this is a process that must operate successfully over decades. On the other hand, any deficits in polarity maintenance have the potential to fundamentally disrupt brain function.

For these reasons, the goal of our proposal is to discover novel mechanisms that neurons use to maintain their polarity. We will do this by taking advantage of a unique case of natural variability. Whilst almost all mature neurons in the mammalian brain possess one, and only one axon, our laboratory has recently identified a population of cells that is very different. In the olfactory bulb, the first region of the brain to process information about the sense of smell, there is a set of neurons that naturally come in different polarity types. Some of these dopamine-releasing neurons possess an axon, but others lack an axon entirely. We will exploit this natural heterogeneity by using cutting-edge technology to compare and manipulate these different sub-types of dopaminergic cell at the molecular level. In doing so, we will ask which mechanisms are crucial for maintaining axons as axons and which are vital for maintaining dendrites as dendrites.

By addressing these important basic biological questions, our proposal has the potential to positively impact future healthcare in the UK. Our study of the olfactory system could benefit future approaches to treat debilitating smell disorders such as anosmia and hyposmia, which affect at least 20% of the population and have a significant impact on quality of life. Our focus on dopaminergic neurons may inform treatments for disorders where these cells are lost in later life, such as Parkinson's Disease. Finally, any new knowledge we generate about ways to maintain the identity of neuronal compartments could prove crucial in therapeutic efforts to repair damage after brain injury or neurodegenerative disease.

Neuronal polarity is vital for the healthy functioning of the nervous system. Using polar features such as axons and dendrites, neurons can compartmentalise and control the flow of information through brain circuits. Neurons are long-lived cells, so this spatial subdivision must be maintained over years to keep network information processing stable. However, while our understanding of the molecular mechanisms driving the establishment of axodendritic neuronal polarity is rather advanced, we know much less about the processes maintaining that polarity thereafter. How do axons stay axonal, and dendrites stay dendritic? Our project will address this fundamental question by exploiting natural variability in a special neuronal type. In the mouse olfactory bulb, dopaminergic interneurons are unusual because their polarity varies from cell to cell. Some have dendrites and at least one axon. The majority, however, are entirely anaxonic. Harnessing this variability, we will contrast axon-bearing and anaxonic cell types at the molecular level to uncover new factors essential for maintaining particular polar states. Our first goal will be a transcriptome-wide comparison of cell types with distinct mature polarity, using single-cell RNA sequencing and targeted bioinformatic analyses that include measures of alternative splicing. We will then localise the mRNA and protein distribution of selected targets in situ, before using cutting-edge CRISPR/dCas9-based techniques to selectively manipulate expression of key molecules. In this way we aim to first identify, then localise, then test the contribution of novel molecular factors to the maintenance of axodendritic neuronal polarity. In so doing, we hope to gain new insight into an underexplored yet fundamental biological process, insight which could prove vital in future attempts to repair the diseased or damaged nervous system.

The proposed project is primarily one of basic scientific research. It promises to make significant academic impact across a broad section of contemporary neuroscience. However, the data, results and knowledge we will produce also have the potential for economic and societal impact for a diverse set of beneficiaries. These beneficiaries can be divided into two broad groups: 1) public sector, commercial enterprises and policy makers with a stake in the provision of healthcare, and 2) the general public.

1) The proposed research has the potential to contribute to the nation's health and wellbeing, directly meeting the BBSRC's Key Strategic Research Priority of 'Bioscience for Health'. By aiming to find new molecules involved in maintaining brain cell structure and function - a process that must operate over decades in humans - we directly address the Council's specific priority of 'Healthy ageing across the lifecourse'. By focusing on the unique properties of newborn cells in old circuits, our work directly impacts on therapeutic attempts to repair damaged or diseased brain tissue through the replacement of newly-generated neurons. In particular, new knowledge of molecules that are essential for keeping neuronal structure intact will be especially important for efforts to re-grow and then maintain previously degenerated or injured pathways in the central nervous system. And, as well as providing basic knowledge which can then be applied to a broad range of issues surrounding normal ageing and mental health, the proposed work will also impact on research aiming to treat specific disorders. Our study of dopaminergic neuron diversity, structure and function may influence attempts to treat disorders based on dopaminergic cell malfunction such as Parkinson's Disease, while novel molecular insight into the olfactory system could impact on attempts to improve recovery rates and quality of life in the significant proportion of the population - 20%, rising to >50% in over-75s - that suffers from debilitating smell dysfunction. Finally, the potential benefits for public health produced by the proposed project can also lead to advances in evidence-based policy making, if, for example, eventual effective treatments can be included in NICE guidelines. In the more immediate term, we additionally hope that our findings can contribute to efforts to persuade policy makers that basic bioscience research is a just, important, and profitable use of public funds.

2) Our research also has the potential to benefit the UK public, by generating openly-available novel data on brain structure and function, and by public engagement in our discoveries. The brain is a fascinating organ, and our research into its inner workings could benefit, amongst others, school students deciding where to take their careers, patient and carer groups interested in the implications for particular disorders, and adults with well-honed scientific curiosity.