L-Aspartate signalling in the brain

Neurons communicate at synapses via the release of excitatory and inhibitory neurotransmitters. The amino acid L-glutamate has long been accepted as the brain's universal excitatory neurotransmitter. A very similar amino acid, L-aspartate, is also present at the synapses of some neurons. Despite L-aspartate being able to activate just one of the suite of receptors that L-glutamate acts on, there is some evidence that it plays a signalling role in the brain. Although neurons are classified as either excitatory or inhibitory depending on which neurotransmitter they release, L-aspartate (excitatory) is found in the synapses of inhibitory interneurons, suggesting that some inhibitory interneurons could release L-aspartate to produce additional excitatory actions.

Our invention of a biosensor for L-aspartate has enabled us to demonstrate the regulated release of L-aspartate in response to stimulation of synaptic pathways in the hippocampus. In exploring the mechanisms that control extracellular L-aspartate, we blocked an intracellular enzyme, asparagine synthetase (ASNS) that converts L-aspartate to L-asparagine. We found that this enhanced the L-aspartate biosensor signals indicating that ASNS, although intracellular, is part of a pathway that regulates extracellular levels of L-aspartate. However, we also found something completely unexpected: during blockade of ASNS the brain tissue started to produce electrical seizure-like activity, reminiscent of the aberrant activity that occurs during epilepsy. This observation is highly significant: human mutations of ASNS that affect its enzymatic activity lead to microcephaly, cognitive impairment, and epilepsy. We have additionally found that ASNS expression is upregulated in brain tissue of human epilepsy patients and in mouse models of chronic epilepsy. We propose that upregulation of ASNS in the epileptic brain is a defensive mechanism that reduces L-aspartate signalling and the incidence of seizure activity.

To advance further our studies, we will identify the neurons that release L-aspartate. We hypothesize that these are the inhibitory interneurons and shall use modern genetic methods combined with L-aspartate biosensing to definitively identify these neurons and explore the key molecular components required for synaptic release of L-aspartate. We shall then determine the neural targets of L-aspartate -the cells it acts on and the actions it has on them. Our final aim has three parts. Firstly, we shall elucidate which cells express ASNS and, when ASNS is upregulated during chronic epilepsy, whether additional cell types express this enzyme. Secondly, we shall test whether upregulation of ASNS is protective, reducing seizure activity. If this is the case, we expect that the epileptic brain (where ASNS is upregulated) will be even more sensitive to inhibition of ASNS than the control brain. Thirdly, we shall genetically delete the expression of ASNS in part of the hippocampus and test whether this makes the brain more likely to generate seizures. Finally, we shall extend our studies to the human condition. By using brain tissue from epileptic patients (removed to help control their epilepsy), we shall test the importance of ASNS in regulating seizure activity and examine the expression levels of ASNS and other key molecular components that we identify in the L-aspartate signalling pathway.

There are ~600,000 people in the UK living with a diagnosis of epilepsy. In 30% of these people, the seizures are not controlled by current medication. Our work to understand, at a fundamental level, L-aspartate signalling in the brain opens a new area to search for additional treatments for epilepsy based around the molecular components of L-aspartate signalling. Dysregulation of L-aspartate signalling is also likely to be important in other neurological contexts such as migraine, stroke and traumatic brain injury.

By developing a biosensor selective for L-Asp over L-Glu, we have demonstrated electrically evoked, Ca2+ dependent release of L-Asp in the hippocampus. The L-Asp signal can be enhanced via blockade of either excitatory amino acid transporters or the intracellular enzyme asparagine synthetase (ASNS). Remarkably, inhibition of ASNS evokes seizure like activity, and we find that this enzyme is upregulated in tissue from human epilepsy patients. Our results suggest that L-Asp signalling, although subordinate to that of L-Glu, may be an important regulator of the excitability of hippocampal networks.

We shall use a combination of electrochemical biosensing, electrophysiology, Ca2+ imaging, chemogenetics and targeted deletion of key genes to tackle the following aims:

1. To identify the cells that release L-Asp. Given that L-Asp colocalises with GABA at synaptic terminals, these are likely to be GABAergic interneurons and we shall identify which subtype is in involved.

2. To determine the postysynaptic targets of L-Asp. We shall screen, via imaging and whole cell patch clamp recordings, the cells that directly respond to release of endogenous L-Asp via NMDA receptor activation and document their patterns of activation.

3. To unequivocally link ASNS to the control of seizures. We shall identify precisely the cell types that express ASNS and whether this pattern changes in the epileptic brain. We shall test whether focal knockout of ASNS in hippocampus enhances seizure generation. We shall explore the functional consequences of the upregulation of ASNS in the epileptic brain. By using resected tissue from human epileptic patients, we shall demonstrate relevance to the human condition.