Beyond The Basal Ganglia: Spinal Circuit Dysfunction As A Final Pathway For Movement Disorganisation In Tor1a Dystonia

Dystonia is a neurological syndrome manifested largely by abnormal movements. It is the third-most common movement disorder, affecting nearly 70,000 people across the U.K. While the causes are varied, the central issue is shared: people have excessive and involuntary, disorganised muscle activity that impairs movement and significantly impacts quality of life.

Dystonia has been considered to be a disorder of the basal ganglia, small nests of nerve cells lying deep in the brain that participate in the selection of movement. In recent years, this concept has broadened to implicate involvement of multiple brain areas. Dystonia is now thought to be a network disorder where dysfunction both within and between these centres leads to movement disorganisation.

The spinal cord is ultimately responsible for organising movement. For example, to flex one's elbow, the spinal cord ensures that the biceps are activated whilst the triceps are inactivated. Given that dystonia is a disorder of movement organisation in which, for example, muscles such as biceps and triceps are activated at the same time, we now seek to determine the role that spinal cord dysfunction plays in dystonia.

Our research objective is to determine the degree to which dysfunctional spinal circuits contribute to movement disorganisation in dystonia. We propose to use an animal model of early onset generalized dystonia (DYT1), one of the most severe and most prevalent forms of genetic dystonia in which signs usually manifest during early childhood initially in the lower extremities and then generalise to the trunk and upper extremities. Using advanced mouse genetics, we will restrict the deletion of the DYT1-related gene (Tor1a) to spinal cord circuity, leaving the brain intact. Specifically, we will manipulate Tor1a expression in all spinal neurons (Aim 1) to determine the role of spinal circuit dysfunction in movement disorganisation in DYT1 dystonia. Next, we will restrict the Tor1a deletion to a population of spinal inhibitory interneurons - nerve cells that collectively prevent muscle co-contractions and that reduce motoneuron/muscle activity, two features that are impaired in dystonia (Aim 2). We will use an array of techniques to quantify the deficits produced by these genetic manipulations.

At the end of this 3-year proposal we will have a solid understanding of the degree to which spinal circuit dysfunction contributes to movement disorganisation in Dyt1 dystonia. These data are crucial for linking dysfunction within upstream regions for motor planning (i.e. the brain) with dysfunction in the downstream organiser of movement (i.e. the spinal cord). In addition, we will have gained insight into the role of a particular class of spinal neurons in the manifestation of signs and symptoms of DYT1 dystonia - a class of neurons that are genetically accessible and thus could be targets for future treatments. As current treatment options are limited, identification of new targets for symptom-alleviating therapies could lead to new treatments aimed at improving movement and quality of life of people with dystonia.

DYT1 is a genetic and severe form of primary torsional dystonia characterised by involuntary, excessive, disorganised muscle activity caused by a mutation in TOR1A. Current hypotheses localise dysfunction to brain circuits. Yet it is the spinal cord that is the CNS structure that ensures movements are organised to produce a behaviour. We therefore hypothesise that spinal cord pathophysiology directly leads to the phenotype of dystonia.

We will perform site- and cell-type specific manipulations of Tor1a expression to address two specific aims: (1) determine the degree to which dysfunctional spinal circuits contribute to movement disorganisation in Dyt1 dystonia; and (2) determine whether dysfunction of a class of spinal inhibitory interneurons contributes to the pathophysiology of this dystonia.

To address these aims, we have developed a new model of Dyt1: a conditional ready model of Tor1a in which exons 3-5 are flanked by FlpO-sensitive target sites. Cdx2-FlpO mice will be used to restrict Tor1a deletions to the spinal cord, sparing Tor1a function in the brain. Using molecular, electrophysiological (in vivo and in vitro), and behavioural techniques, we will quantify the degree to which spinal cord circuits contribute to the phenotype. We will compare these findings with those obtained through another new model in which Tor1a will be spared in the spinal cord and deleted in the brain. Next, we will use intersectional FlpO-Cre deletion of Tor1a-frt/flox to combine site- (spinal) and cell-type specificity, targeting a class of spinal inhibitory neurons with bi-allelic Tor1a deletion.

By the end of this proposal we will have a solid understanding of how spinal circuit dysfunction contributes to movement disorganisation in Tor1a dystonia. This understanding could lay the foundation for possible new targeted therapies to treat signs and symptoms in order to improve the quality of life of people with this challenging disease.

Neurological diseases are costly for all societies. In high income countries, they are costly to treat, and in low and middle income countries, they are often too costly to treat and result in stigmatisation and premature loss of life. Thus it is clear that we have a responsibility to create the knowledge that will ultimately lead to improved treatments, which will ultimately lower costs. (Note that there is always a lag, with new treatments initially associated with higher costs that ultimately lessen, leading to cost reductions for the treatment of the disease - see Pardes et al, Science 1999, 283:36).

Future treatments of neurological diseases will likely focus on genetic therapies. To develop such treatments for diseases that affect neural circuits (circuitopathies), we need knowledge of the circuits as well as of the genetics of the cells within them, plus knowledge of the circuit pathophysiology. This is the knowledge we aim to create here. With this foundation, new genetic therapies could ultimately be developed, leading to a number of beneficiaries. We have discussed academic beneficiaries above. Other beneficiaries include:

Clinical beneficiaries. We are investigating the contributions of spinal circuit dysfunction to movement disorganisation in DYT1 dystonia. This is a new line of inquiry in dystonia research which may shed light on new neural substrates for therapeutic intervention. Immediate prospective beneficiaries are patients with generalised DYT1 dystonia, their primary carers, and health care workers.

An emerging hypothesis in dystonia research is that multiple forms of dystonia may share a common neural substrate. As such, results from our DYT1-specific model could generalise to other forms of dystonia, leading to additional clinical beneficiaries.

In general, movement disorder research principally focuses on dysfunction within supraspinal centres. By highlighting the contributions of spinal circuit dysfunction to movement disorganisation in dystonia, our data will raise awareness for other movement disorder specialists to consider this often-neglected component of motor control. (As a functional neurosurgeon, RB engages regularly with movement disorder neurologists, and will continue to engage with them regarding new findings.)

Commercial private sector: The long-term goal of this work is to understand the circuitopathy of dystonia such that circuit therapy can be developed to improve quality of life (see Brownstone & Lancelin 2018). While these therapies are not around the corner, we expect that the work described herein could lay the foundation for eventual treatments, leading to beneficiaries in industry. The development of any new therapy will require the involvement of industry. If, for example, we find evidence that En1-expressing spinal neurons may be a potential target for symptom-alleviation in dystonia, then once proof-of-concept studies are successful, the private sector will be needed to move towards first-in-human studies aimed at targeting these neurons.

Training: This proposal will lead to the further training of the co-applicant, post-doctoral research associate AP, who will be at the forefront of a new direction in the field of dystonia. Amanda has developed new skills already in the lab, for example those related to mouse genetics, disease models, and electrophysiology. We will also train a research assistant with these and other new skills. These young investigators will sharpen their scientific cognitive skills as well, and will be ready to be future leaders in or out of academia.

We also plan to engage in public events when possible, in order to increase understanding of science. We will provide opportunities to young talented school children to experience scientific research first hand through pairing schemes.