The mechanical control of neuronal maturation

During the development of the nervous system, billions of neurons have to extend long processes (dendrites and axons), which grow over large distances, become electrically active, connect to the right partners and communicate with them. Through these connections neurons form highly organised networks and transmit information in form of electrical and chemical signals that govern our functions. Each of these developmental steps is critical, and any failure may have devastating consequences for the whole organism.

Almost everything we know about these processes is related to the communication between neurons and their environment via molecules and electrical signals, which is what biology has focused on during the past decades. However, neurons live in a physical world and obey physical laws. When neurons grow through tissue, they not only chemically but also mechanically interact with their environment. As bicycling is easier for us along a paved road than along a sandy beach, the mechanical properties of the tissue through which neurons grow will also strongly influence, for example, how fast they can grow and develop.

Importantly, the local stiffness of brain tissue varies depending on the region in the brain, and it changes during development, ageing, neurological diseases and after injuries. While brain tissue is usually extremely soft, it becomes stiffer during ageing (in men more than in women), and under pathological conditions it can change dramatically in structure and stiffness. Prominent examples are scarring after injury or stroke, and the formation of rigid plaques or tangles in diseases such as Alzheimer's. These changes in local tissue stiffness may strongly influence neuronal function. Neuronal function, which develops as neurons mature, is characterised by their capability to generate and transmit electrical signals. However, how the mechanical environment regulates the maturation of the electrical activity of neurons and their connections to other neurons (synapses) is not known.

To address this important gap in our knowledge, which has important implications not only for the development of the nervous system but also for different neurological disorders, we have put together a multidisciplinary team with years of experience in neuroscience and biophysics. The proposed project involves cutting edge neurobiology, mechanobiology, electrophysiology, molecular biology, biophysics and engineering approaches, whose combination will advance the field and provide powerful tools beyond the state of the art.

We will first determine which mechanical properties the environment must have to optimally promote neuronal maturation and activity, by comparing how neurons develop in mechanically different custom-built environments. We will then use a tiny leaf spring ('cantilever') to push and pull on neurons with well-controlled forces, which are as small as the forces cells usually exert on their environment, and simultaneously measure the electrical currents that flow through these neurons. These experiments will reveal how mechanical signals alter neuronal activity and maturation. Finally, we want to understand how neurons perceive and translate these mechanical stimuli. To do this, we will identify force sensors in the neurons, and investigate how their specific activation changes cellular function.

The knowledge gained in this project will not only illuminate a new facet of the development of the nervous system. Mechanical signalling might also be the missing link to understanding different developmental disorders and neurological diseases. Our research, bridging the gap between the life and physical sciences, may thus ultimately lead to important changes in how we treat patients suffering of neurological disorders.

During CNS development, neurons migrate, extend processes, connect with their targets and become electrically active. Despite their importance, none of these processes is fully understood. While current knowledge is almost entirely based on molecular and electrical signalling, throughout development neurons also mechanically interact with their environment. However, the impact of mechanical signalling on neuronal maturation is currently unknown.
To fill this important gap, we will combine our strengths in mechanobiology and neurophysiology to investigate the role of mechanical signals in neuronal maturation and underlying mechanotransduction pathways.
By culturing primary hippocampal neurons on custom-built compliant substrates of varying stiffness, we will mimic mechanical signals neurons encounter in the developing CNS. A combination of immunocytochemistry and patch-clamp experiments will reveal how electrical maturation and synapse development depend on substrate stiffness and time in culture. To test how neuronal activity is modulated by acute mechanical stimuli, we will develop a setup combining patch-clamp with optical and atomic force microscopy. We will apply controlled mechanical stresses on neurons and determine dose-response functions for strain-mediated currents. We will furthermore study how tension, to which axons are constantly exposed in vivo, modulates electrical activity and synaptic protein transport. We will then exploit this setup to identify mechanosensitive ion channels regulating neuronal mechanosensitivity, and test their functional relevance using siRNA in cultures on compliant substrates. Ultimately, we will use single-neuron RNA-Seq to investigate potential downstream signalling pathways, and test candidate genes in siRNA experiments.
The inclusion of mechanics in our picture of CNS development could lead to a paradigm shift in developmental neurobiology and to unexpected breakthroughs in the prevention and treatment of CNS disorders.

The proposed project is highly interdisciplinary in nature. It combines biological, physical, engineering and medical aspects, is concerned with the design of novel techniques and investigates molecular mechanisms potentially relevant to development, pathology and medical treatment. Thus, there is a wide range of direct and indirect beneficiaries from the research, including commercial partners, the general public and the researchers themselves.

Potential commercial partners include the microscopy and biotechnology industries. We will introduce new ways to use atomic force microscopy (AFM) and electrophysiology that will generate new platforms and applications. Potential partners will be interested in securing/licensing IP arising from this research to market products for the research sector; we have established contacts within JPK (AFM) and Zeiss (microscopy) who will be interested in using our techniques to widen the scope of potential buyers. We will ensure the longest possible exploitable patent exclusivity; patents will directly emerge from the research towards the end of the project duration. We will directly interface with possible industry partners to secure commercialisation of the developed techniques. This direct, short- to mid-term impact will be a strengthening of the competitiveness of the microscopy industry in the UK.

Understanding how cells respond to their mechanical environment and establishing methodologies/materials that enable directing cellular responses will be of enormous benefit for biotechnology research and industry, particularly for tissue engineering. We expect a high potential impact in the biotechnology area and will actively search for relevant systems/companies to share our knowledge. The impact will be direct and midterm.

Unravelling how mechanical signalling is involved in controlling neuronal maturation and functioning will also provide a starting point for the development of pharmaceutical products influencing cellular responses to mechanical stimuli and thus potentially even cellular ageing. Providing appropriate signals to neurons to regain functionality might help overcoming their failing regeneration after injuries, or even improve the clinical picture in neurodegenerative diseases. Thus, there is a great potential of commercialising products used to regain neuronal function. It will be direct and mid- to long-term.

Interdisciplinary research, and the breaking down of scientific boundaries, is a hot topic that excites the general public, who we will engage through talks aimed at larger audiences. Moreover, contributing to the successful treatment of neural disorders has an enormous impact on general health. Treating spinal cord injuries or neurodegenerative diseases will improve the quality of life of thousands of people in the UK and beyond the borders. Furthermore, it will drastically reduce treatment costs, thus directly and indirectly impacting the healthcare system. The impact is indirect and mid- to long-term.

Understanding cellular responses to mechanical cues is highly relevant to biology and biophysics. It is known that mechanosensitivity is involved in many physiological and pathological processes ranging from embryo formation to liver cirrhosis, adding an impact on medical research. The development of novel methods is particularly relevant to engineers. Accordingly, scientists working in any of those areas might be highly interested in the outcome of the project. The impact will be direct and immediate.

Staff involved in the project will work interdisciplinary, interact with many scientists of different backgrounds and companies and creatively solve problems. They will further develop communication, problem solving and entrepreneurial skills and acquire new technical and IT skills, which will make all involved ultimately more marketable in the employment sector, from physics and biotechnology to regenerative medicine. This will generate short-term impact.