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Modulation of Basket cell microcircuits in cerebellar cortex paroxysmal ataxia disorders

Periodic Reporting for period 1 - BaskAtax (Modulation of Basket cell microcircuits in cerebellar cortex paroxysmal ataxia disorders)

Reporting period: 2017-10-01 to 2019-09-30

Episodic neurological disorders such as migraine and epilepsy are common, yet in most cases our understanding of their origins and causes remains surprisingly basic. This in turn has hampered the development of targeted therapy for the treatment or relief of symptoms. Typically these disorders have a complex genetic pattern, nevertheless a small subgroup have a simple monogenetic origin. These rare monogenetic episodic disorders can individually affect many genes, however there is a bias towards genes linked to neurotransmission and particularly the function of the synapse the point of communication between neurons in the brain.

We have studied a rare congenital neurological disorder called Episodic Ataxia Type 1 (EA1) caused by a dominant negative mutation in the Kcna1 potassium channel. Individuals with EA1 suffer from muscle twitches (myokymia) and paroxysmal events lasting from minutes to hours, characterized by loss of coordinated movement (ataxia). The cerebellum, a brain region often affected in ataxia is enriched with synaptic Kcna1 potassium channels. Consequently it is thought that EA1 mutation in Kcna1 has a pathological role in cerebellar synaptic neurotransmission. In this project it has been our objective to understand the effect of Kcna1 mutation on the function of cerebellum and to investigate potential new treatments. These experiments are not only important to individuals living with EA1, but given the crossover with other synaptic disorders has the potential to identify communalities that can be applied to the class of episodic neurological disorders as a whole.

To address these objectives we have recorded the function of the cerebellum in a mouse model of EA1. Contrary to expectations we discovered that the baseline function of the cerebellum was not affected in EA1. This was supported by behavioural assay showing that motor function was retained in these mice. We interpret this to indicate that robust neuronal mechanisms function to normalize cerebellar function in EA1. However, in contrast when we challenged EA1 mice with stimulants aping a paroxysmal event differences became apparent. Using a new behavioural paradigm, we also made the unexpected discovery that male and female EA1 mice are differentially affected. Our experiments suggest that adaptation has an important role in protecting the brain in episodic neurological disorders and that how males and females respond to paroxysmal attack triggers is not necessarily the same.
To understand the effect of EA1 on cerebellum function, our experimentation required us to make electrical recordings from groups of neurons in the cerebellar cortex. These technologically challenging experiments allowed us to measure the precise temporal activity of groups of neurons as signals passed through the cerebellum. Basket cells are known to signal to Purkinje cells exceptionally quickly via a process called ephaptic transmission. We had hypothesized that ephaptic transmission might be compromised in EA1 due to the requirement of Kcna1 potassium channels.

- We found that in contrast to expectations ephaptic neurotransmission was not affected in the EA1 cerebellum.
- We used confocal microscopy to image points of ephatic neurotransmission. On average EA1 connections were not significantly different from controls.
- Consistent with a compensation for Kcna1 mutation, Purkinje cell output from the cerebellum was normalized in adult EA1.

We created a new technique using the expression of fluorescent molecules that allowed us to visualize individual synapses while simultaneously recording electrical activity. Using this method we were able to measure for the first time the relative strength of individual connections between Basket and Purkinje cells.

- We found that connections were highly heterogeneous. Most Basket cells alone produced only a modest change in Purkinje cell electrical activity, however a subset of 'super' Basket cells individually caused a large and extended pause in Purkinje cell activity.

To understand the effect of Kcna1 mutation on behaviour we challenged adult mice harbouring the Kcna1 mutation with tests of motor coordination. Further, since paroxysmal events in EA1 individuals can be precipitated by stimulants, we also exposed the EA1 mice to mild stimulants looking for periods of episodic ataxia.

- Baseline motor coordination in EA1 mice was not affected. This finding agrees with our electrical measurements of cerebellum output that indicated adaptation to the Kcna1 mutation.
- Unexpectedly, we found a motor phenotype in response to stimulants that was only present in female Kcna1 mutant mice.

Current therapeutics for EA1 are limited. The discovery of a motor phenotype in a model of EA1 offers the platform for the trialling of new treatments. This is currently being exploited with a collaborator investigating the suitability of exon replacement gene therapy in EA1.

The results from this project have already been shared at conference presentations including: Synaptopathies 2018, UCL Neuroscience Symposium 2019. The results will also be imminently published reaching a broad scientific audience. The project has been especially fortunate to also disseminate results with individuals directly affected by ataxia, and a summary of the findings will be published in the spring 2020 issue of Ataxia Magazine.
These results not only advance our understanding of the cerebellum, but also how this brain region responds to pathological mutation. This project also describes for the first time how ephaptic neurotransmission is protected in response to genetic perturbation.

- Ephaptic signalling has been underappreciated. This study for the first time describes the accommodation of ephaptic signalling in a contemporary model of a neurological disorder. We found that ephaptic signalling is surprizing robust, pointing to its relative importance in neuronal function. These findings will highlight the importance of ephaptic signalling and impact future studies of the cerebellum.
- This project has identified a new behavioural phenotype in a model of EA1. Exploitation of this model as a platform for new therapeutics has the possibility of directly impacting patient groups living EA1. Already with collaborators we are trialling gene therapy with this new assay.
- The unanticipated increased susceptibility of females to the behavioural paradigm introduces a role for gender in EA1 for the first time. Gender is known to have an important role in other episodic disorders such as migraine. It is anticipated that this finding will have the impact of encouraging clinicians to examine a role for gender in episodic ataxias.

The innovative and technologically advance methods employed in the project also advance the field of electrical study of the brain. These techniques will impact experimental neuroscientists and potentially open new avenues to study.

- The project has identified an optimized method to record from the adult cerebellum. These methods advance the state of the art in electrical recordings from difficult brain structures.
- A novel method for the sparse fluoresecent and opsin based labelling of Basket cells was developed in this project. This for the first time allows targeted recording of Basket cell - Purkinje cell populations and advances the state of the art of cerebellar investigations.
Basket cell (red) synapsing onto multiple Purkinje cells (green)