HSN1 secondary to SPTLC1/2 mutations: Pathogenesis and treatment

Lead Research Organisation: University College London
Department Name: Institute of Neurology

Abstract

Hereditary Sensory Neuropathy Type1 (HSN1) is a rare inherited disease affecting the nerves in the arms and legs responsible for sensation and movement. The initial symptoms are loss of pain and temperature sensation and numbness in the feet. This slowly progresses with variable but often severe weakness in arms and legs leading to significant disability. There are frequent serious complications including amputations. As yet, there is no treatment available.
HSN1 is caused by mistakes in the genetic code (mutations) of the genes SPTLC1/2. Recent studies have shown that mutations in these 2 genes causes an alteration in the cellular reaction which leads to the build-up of abnormal compounds which are called deoxysphingolipids (DSBs) which cause nerve damage. However, the evidence for DSBs being toxic to nerves is not conclusive and the exact mechanism of how they cause nerve damage is not known.
A recent study has showed a possible treatment option using serine, one of the building blocks of proteins. Treatment of mice harbouring the disease causing mutation with serine, led to an improvement in function. A pilot HSN1 study in patients showed that serine was well tolerated however it was too short in duration to assess if the treatment was beneficial. This has led to the possibility of a treatment trial using serine in HSN1 patients in UK.
The aim of this project is to answer two fundamental questions which will provide strong support for this trial.

1)Are DSBs toxic to nerves and can treatment with serine reduce this toxic effect?
To answer this question, several series of experiments will be performed tackling the question from different angles. Firstly, nerve cells from mice will be used. The toxic effects of DSBs will be assessed by looking at the effects on nerve survival and growth when the nerves are grown in solutions containing DSBs. To look at the therapeutic effects of serine treatment, cell survival and growth will again be assessed but this time with the nerves grown in a solution supplemented with serine. Further experiments will also be performed, investigating how DSBs cause nerve damage.
The second set of experiments involves using skin cells obtained from HSN1 patients and reprogramming them into stem cells (cells with the potential to grow into any cell type in the body). These cells are then directed to grow into nerve cells. The therapeutic effect of serine can be tested by comparing cell survival and growth between the serine treated and untreated human cells. These cells can also be used to probe further into what causes nerve damage in HSN1.

2) What is best way of assessing disease progression which can be used in a trial to assess whether serine treatment is beneficial?
The severity HSN1 varies significantly between patients. A variety of investigations (clinical examination, MRI of calves and thighs, machine muscle strength testing, skin biopsy, blood tests, electrical tests of nerve function in arms and legs and assessments of different sensations for example hot and cold detection) will be performed in order to capture the spectrum of deficits seen in this condition .These will be repeated after a year and the results will be analysed to determine the best method of measuring disease progression.

By the end of the project, the therapeutic extent of serine treatment will be known. Using different laboratory set ups as described above to evaluate this will increase the validity of the findings. Finding ways to measure disease progression in this disease will clear the final hurdle on the path to a therapeutic treatment trial with serine.

Technical Summary

1)Determining deoxysphingolipid (DSB) induced neurotoxicity and evaluating L-serine supplementation as a therapeutic option.
a)Mammalian in-vitro model (primary motor neuron cultures from wild-type mouse embryos and primary dorsal root cultures from wild type mouse pups): These will be used to determine toxicity by assessing the effects on neuronal survival and neurite outgrowth following treatment with DSBs. Co-treatment with L-serine will then be evaluated to determine if it is neuroprotective. Further studies will look at differential toxicity to DSBs between different sensory subpopulations and evaluate whether there is differential uptake of fluorescently labelled DSBs using confocal microscopy. Cellular pathomechanisms will also be explored, initially looking at whether TRPA1 receptors are activated by DSBs.
b)iPSC derived sensory neurons from HSN1 patients' fibroblasts: At first, the iPSCs will be differentiated into sensory neurons and phenotyped using functional calcium imaging following application of nociceptive agents and thermal stimuli and electrophysiological studies. After establishing a lineage that phenotypically resembles HSN1 (for example by assessing DSB synthesis in these neurons), these cultures acting as patient specific in-vitro disease models, will be used to assess the therapeutic effects of L-serine supplementation. Later, this model will be used to explore disease mechanisms.

2)Determining responsive outcome measures.
HSN1 patients will undergo a set of investigations which will be repeated after a year's interval. These include: CMT Neuropathy Score, comprehensive neurophysiological assessment, Quantitative Sensory Testing (DFNS protocol), muscle MRI studies of the thighs and calves (T1 weighted, STIR, IDEAL and CPMG sequences), myometry, analysis of plasma DSB levels, upper thigh skin biopsy (epidermal nerve fibre density measurements) and patient questionnaires (SF36 and NPSI)

Planned Impact

Like in other rare diseases, patients with HSN1 are at a disadvantage as evidenced by a delay in diagnosis, lack of evidence based guidelines on management, limited active research in the area and very little pharma involvement.

1) What are the applications of the research relevant to patients and patient organisations?
a. Evidence for the therapeutic effects of L-serine supplementation obtained from in-vitro mammalian models and iPSC derived sensory neuron cultures will provide strong support for a clinical trial of oral L-serine supplementation. The natural history component of the project will determine a method of measuring disease progression in this highly variable condition which can be used as an outcome measure in a clinical trial. The goal of serine treatment is to slow disease progression and reduce the symptoms of neuropathic pain. The disease is slowly progressive but over time causes significant disability due to profound sensory loss and the resultant sensory complications and weakness. This along with the neuropathic pain has a huge detrimental impact on patients' quality of life. The aims of treatment with serine are to improve quality of life, prolong independence and ability to work. This impacts not just the patients and their families but also health and social care services. This would also pave the way for a treatment trial in children, which will maximise the benefit.
b. Better understanding of the clinical phenotype for early diagnosis and counselling of patients.

2) What are the applications of the research which are relevant to academic colleagues?
a. Better understanding of the clinical phenotype will help focus research into the cellular pathomechanisms.
b. The method for measuring disease progression can be adapted and applied by researchers investing other rare heterogeneous diseases.
c. Investigating a possible common pathogenic pathway between HSN1 and diabetic neuropathy involving sphingolipids. With increasing incidence of diabetes, this research will be important to both patients and health care services.

3) What are the applications of the research which are relevant to Industrial Partners?
a. The clinical and laboratory based aspects of the project will provide support for undertaking a clinical therapeutic trial using oral L-serine supplementation in the next 3-4 years.
b. Establishment of an iPSC derived sensory lineage that models HSN1 will enable large scale studies of small molecule libraries with the potential for both biological insights and therapeutics.
c. Better understanding of deoxysphingolipid induced neurotoxicity might provide other targets for treatment and trial of known agents involved in the early sphingolipid biosynthetic pathway.
d. Fostering close links between the Pharmaceutical industry and MRC centre, building on the established set-up for carrying out national and international clinical trials.

4) What are the applications of the research which are relevant to policy makers?
a. Establishing a national centre specialising in the treatment of HSN1 and providing patients with access to specifically trained allied health professionals.
b. Unique training and research opportunities for neurology and clinical neurophysiology trainees and allied health professionals (physiotherapists, occupational therapists, orthotics department) wishing to sub-specialise in this area. This extends to people beyond the UK borders.

Publications

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Title Human iPSC derived model of HSN1 
Description Human iPSCs derived from HSN1 patient fibroblasts which were then differentiated into sensory neurons. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2017 
Provided To Others? Yes  
Impact 1) Further investigate the pathomechanism underlying this disease 2) Human Model to test the therapeutic effects of serine and other potential therapeutic agents for HSN1, 
 
Title England HSN1 Database 
Description Database of patients with the rare condition, Hereditary Sensory Neuropathy Type I in England 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Target patient population already identified for potential therapeutic clinical trials 
 
Description Sensory neurons derived from iPSCs from HSN1 patients 
Organisation Oxford University Hospitals NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution Provision of fibroblasts from Hereditary Sensory Neuropathy type 1 patients to expand the collection currently held at the Institute. The fellowship also provides a research student to phenotype the sensory neurons and lays the foundation for further work
Collaborator Contribution Providing a lab with expertise in differentiating sensory neurons from iPSCs and also in the function assessment of sensory neurons
Impact Collaboration commenced in February 2016
Start Year 2016