The Pathogenesis of Spinal and Bulbar Muscular Atrophy

Spinal and Bulbar Muscular Atrophy (SBMA) is a rare form of adult motor neuron disease. It leads to slowly progressive weakness in the muscles used for speech and swallowing as well as in the arms, legs and face. Patients usually first start noticing symptoms between the ages of 30 and 50 with cramps and mild weakness in their legs but they can end up needing a wheelchair, having problems with speaking and swallowing drinks and requiring full-time care.

SBMA is rare and it is caused by a problem in the androgen receptor on the X Chromosome. The disease affects males and occurs when the faulty androgen receptor binds to testosterone the male sex hormone. There is no cure for SBMA and there are currently no treatments available for this disease.

Motor neurons are large nerve cells with long processes that extend from the spinal cord to the muscles and carry the messages to make the muscles contract. In SBMA the motor neurons become dysfunctional and eventually die, this is called neurodegeneration. It is not yet understood exactly how this genetic problem leads to the motor neurons dying. Although SBMA is a rare disease, it is particularly interesting as it has many similarities to other neurodegenerative diseases including the aggressive form of motor neuron disease (MND) and Huntington's Disease. Looking at how the motor neurons die in SBMA may also provide insights into how neurodegeneration occurs in these other conditions.

I am going to look at possible pathways leading to neurodegeneration in SBMA. These pathways have been identified from previous research in SBMA and other neurodegenerative diseases.

1. Axonal transport - axons are like the motorways of the cell carrying the messages between the cell body which is the control centre of the cell and the synapses where the cell communicates with other cells. Motor neurons have very long axons and so are at risk if there is a problem with the transport (like a traffic jam on the motorway).

2. Protein handling - this is how the cells cope with stress. A series of protective steps usually happen in a cell in response to stress (heat, lack of oxygen, too much protein) however in SBMA there is some evidence that motor neurons do not cope with stress as well as a normal motor neuron.

3. Endoplasmic reticulum stress - the endoplasmic reticulum (ER) is important for making new proteins in a cell. In SBMA the protein is abnormal because of the genetic mutation. This can lead to stress of the ER as it tries to cope. If the ER becomes overloaded it triggers a mechanism that causes the cell to die.

4. Mitochondria - these are like the batteries of the cell and provide most of the energy needed for all the things that cells do. If there is a problem with the mitochondria, this will affect the energy levels of the cell and may lead to the cell dying.

5. Astrocytes - these cells provide support to motor neurons and communicate with them. In motor neuron disease it has been shown that problems in the astrocytes, and how they speak to the motor neurons, can lead to motor neuron death. This has not been studied in SBMA yet.

So far, research in SBMA has been done in cell and animal models of SBMA and this means that the findings may not actually be applicable to human patients. Recent advances in science have led to a technique which makes stem cells (cells which can turn into any human cell) from patients' skin cells. These cells are called induced pluripotent stem cells (iPSCs). For this project, I will grow the iPSCs into motor neurons and therefore I will be able to look at human motor neurons "in a dish".

In summary, this project will investigate some of the mechanisms that have been suggested to cause motor neuron degeneration in SBMA using motor neurons "in a dish" which have been created from patients cells. As there is no cure for SBMA, one of the main aims of this project is to identify potential targets for drug treatments.

All the experimental protocols described are established in my host labs

0-18m: Characterise iPSCMNs
Initially 2 patient, 2 healthy control lines. More lines are available
Differentiate MN: Spinal: Developmentally rationalised cues used including retinoic acid (RA) and Sonic Hedghog (SHH). Bulbar: WNT agonist combined with RA for rostrocaudal patterning. MN creation: Immunocytochemistry (HB9, ISL1, SMI32, ChAT), electrophysiology
SBMA: karyotype analysis, CAG using Sanger sequencing, expression and localisation of AR
Cell death: cell counts +/- dihydrotestosterone by immunofluorescent staining using Dapi, B-III tubulin and actived caspase 3 (quantified with Western Blot)
Axonal transport: Live cell imaging and a fluorescent atoxic tetanus toxin fragment for retrograde AT, Mitotracker for anterograde/retrograde mitochondrial AT
Protein mishandling: WB and immunostaining for components of heat shock response (HSR) at baseline and following exposure to cell stressors
ER stress: Changes in cytosolic Ca2+ levels using ER stressors (thapsigargin, ionomycin) and fura-2. Markers (PDI, CHOP, BiP ATF4) with WB and immunostaining
Mitochondria: Live cell imaging with fluorophores (TMRM, FCCP, Rhod 5N, Indo-1 AM) to measure: membrane potential, redox state, and Ca2+ levels. Electron microscopy to assess morphology

0-24m: Isogenic controls
CRISPR spacers to replace expanded CAG repeat with normal length sequence in a patient line and insert an expanded repeat into a control line. Confirm genetic correction with PCR

18- 36m: Astrocyte-neuronal interaction
Differentiate astrocytes: Precursors cycle in FGF2 for >80d then terminal differentiation with BMP4 and LIF
Phenotype: Reactive state using process length, GDNF and GFAP upregulation. Co-cultures of iPSC-astrocytes and MNs, to examine Ca2+ regulation and mitochondria function

28-36m: Therapeutics
Test potential therapeutics which may ameliorate MN dysfunction as described in the experiments above

Impact
For patients and patient organisations:
- This research may provide relevant insights for the development of mechanism-targeted therapeutic agents
- A therapeutic drug would improve/prevent progression of disability in patients and hopefully improve quality of life.

For the wider public
- This research could contribute significantly to the much wider field of neurodegeneration.
- Identifying common pathways in cell death may suggest potential therapeutic targets to ameliorate other neurodegenerative diseases.

To academic colleagues
- Developing motor neurons derived from iPSCs will provide an invaluable model to researchers in Prof Greensmith's lab investigating other aspects of SBMA pathology.
- Characterising axonal transport and features of bulbar motor neurons in iPSCs will be of value to other iPSC researchers.

To commercial and private sector beneficiaries
- Understanding of mechanisms and causes of motor neuron loss could lead to identification of potential therapeutic targets which would be of significant interest to pharmaceutical companies.
- A model to screen drugs which may impact motor neuron survival in this disease would also be beneficial to drug development.

For policy makers
- Better understanding of neurodegenerative mechanisms and identifying therapeutic drugs which ameliorate these processes has significant implications and benefits for policy makers by potentially decreasing the burden of disease, increasing the quality of life and functional independence of patients with SBMA and hopefully other neurodegenerative conditions.