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Food for thought: monitoring the effects of drugs and diet on neuronal glutamate release using nanoelectrodes

Periodic Reporting for period 1 - F4TGLUT (Food for thought: monitoring the effects of drugs and diet on neuronal glutamate release using nanoelectrodes)

Reporting period: 2019-05-01 to 2021-04-30

Neurons are the cells in the brain whose main task is transmitting information. They can do so in two ways: electrical, via pores between cells, or chemical, via release of neurotransmitters. The location of this communication is the synapse, a structure at the end of the branch-like structures of neurons. There are many different neurotransmitters with different effects. Glutamate is the primary activating neurotransmitter in the brain. It modulates the strength of connections between neurons, which is understood to underlie memory formation. However, it also plays a role in several diseases of the brain, such as depression, ADHD and addiction. Overstimulation by glutamate can have toxic effects on neurons. This is thought to be involved in diseases characterized by dementia, such as Alzheimer’s and Parkinson’s. With ageing being the most important population trend in large parts of the world today, these diseases are expected to become even more prevalent, and no curative therapies currently exist. Even though many drugs that are used against these diseases have effects on glutamate receptors, it is unclear to which extent glutamate neurotransmission are changed during disease, or how drugs change them. Besides drugs, it is known that certain dietary compounds, such as cholesterol and omega-3, can influence the glutamate signalling system in the brain. To elucidate some of these (changes in) glutamate levels during health and disease, this project aimed to monitor glutamate release in the synaptic cleft between neurons.

For this, electrodes and electrochemical detection were employed. The advantage of this technique is that electrodes can be easily miniaturized, to allow sensitive measurement of very fast events at the cellular level. Unfortunately, glutamate itself cannot be electrochemically detected directly. The project proposed to overcome this by developing a glutamate biosensor. Biosensors employ a biological recognition element – in this case the enzyme glutamate oxidase – which is immobilized on an electrode and specific for the analyte. The enzyme converts the analyte, while producing an electroactive reporter molecule – in this case hydrogen peroxide. The hydrogen peroxide can then be detected at the electrode. The biosensor is based on a nanoscale electrode, which allows measurement in the synaptic cleft. In the project, a novel immobilization method for enzymes was developed. This method allows the creation of very thin layers of enzyme, allowing the sensors to operate at maximum spatial and temporal resolution. This is necessary to be able to resolve the sub-millisecond dynamics of the glutamate release events at neurons. Within the project, the use of human induced stem cells was established in the host lab. These cells can be differentiated into glutamatergic neurons. A microfluidic device was designed, fabricated and used for the culturing of neurons. This can be used to assist the probing of the very small synaptic cleft structure, as neurons can be grown in a controlled way. The project ran for 11 months in the Andrew Ewing lab at the University of Gothenburg, Sweden.
In the project, a number of results were achieved. Nanoscale electrodes that were earlier developed in the host lab were made suitable for use as a biosensor substrate. This was necessary, as the initial electrodes were made of carbon, whereas platinum is more appropriate as a material for electrochemical biosensors. A general immobilization method, which uses a hydrogel coating on the surface of the electrode to fix glutamate oxidase in place, was first pursued. However, it was found that this approach was not suitable for this project: the response time of the sensors was too slow and the range of concentrations that could be reliably measured not wide enough. To ameliorate this, a novel enzyme immobilization technique was developed in collaboration with the German company Nanotag Biotechnologies. This technique employs molecules that can self-assemble into a one-molecule-thick layer on the electrode surface. These molecules are functionalized with specific chemical groups that can “click” to enzymes functionalized with complementary groups. This approach was successfully applied for the immobilization of an enzyme similar to glutamate oxidase, and will be presented in a publication that is currently under preparation. The work was also disseminated at the large analytical chemistry conference Pittcon 2020 (Chicago, IL, USA).

The microfluidic device for the cultivation of neurons and other brain cells was designed, fabricated and validated by growing neuron-like LUHMES cells for several days. However, true human neurons are very complex and difficult to cultivate. Thanks to a standing collaboration between the Ewing lab and the Ernst lab at McGill University (Montreal, Canada) we had access to human, stem cell-derived neurons. Within the project, the necessary infrastructure was established and experience gained to successfully grow these cells. The first results of the electrochemical interrogations performed on these cells will be reported in a manuscript currently in preparation. Finally, the microfluidic platform that was developed at the start of the project was repurposed for a new project that involves the imaging of neurons using a super-resolution STED microscope. This will allow the visual inspection of the biophysical phenomena involved with neurotransmitter release.

No dedicated website for the project was made.
One of the current limitations of biosensors technique is enzyme instability as a result of sub-optimal immobilization methods. Therefore, the novel immobilization method developed in this project could generate potential for the use of biosensors in a wide range of applications. These could be in fundamental scientific projects not dissimilar from this one, as biosensors are especially useful when investigating (sub-)cellular phenomena. However, they could also be used in drug development, to assess the effects of drug candidates.

Another potentially interesting result that could have great societal implications is the use of human stem cell-derived neurons for biological research. These cells can be easily obtained from (adult) humans, healthy or otherwise, as skin cells. Current technology allows us to bring these skin cells back to a pluripotent, stem cell state. These stem cells can then be differentiated into other cell types (such as neurons) using specific culture conditions. Using these cells, scientists can study the fundamental biological properties and functioning of healthy or diseased cells. These cells could become a very interesting alternative to the still wide-spread use of animals in drug development. Effective elimination of unsuccessful candidates using cells at an early stage of the process can reduce the number of animals. Moreover, this would mean that multi-year drug development processes can perhaps be shortened, with higher success rates and lower costs.
Schematic representation of nanoscale glutamate biosensor in synaptic cleft between two neurons