HSP Research

‘Towards a Cure for HSP’ Research Program

 

  • Planning for Clinical Trials 2017

  • Validating Therapeutic Drug Candidates for treating HSP 2014-16

  • Testing & Selecting Therapeutic Drug Candidates for treating HSP  2013-14

  • Identifying Therapeutic Drug Candidates for treating HSP 2011-12

  • Stem Cell Pilot Study 2009-10

Planning for Clinical Trials 2017

With the successful validation of drug candidates completed, the focus of the HSP Research Program is now on clinical trials, which are aimed at determining the extent to which a drug treatment found to be effective with human HSP stem cells in the laboratory is also effective with HSPers themselves. The clinical trials team has met to begin the substantial planning process. Here is an overview of the output from the planning meetings to provide a sense of the scope and scale of the overall initiative and the issues involved.

An update on the HSP Research Program is published every three months. The latest report can always be found in the articles on the homepage.

 

Biomarker(s) For HSP
A biomarker is a measurable indicator of the severity of a disease. Currently there is no measure of HSP that is immediately suitable for use in a clinical trial by which to gauge the effectiveness of any treatment. There is no objective clinical measure of sufficient sensitivity to assess HSP severity or how that changes over time. In order to determine the effectiveness of any treatment for HSP in clinical trials, having a sufficiently sensitive and reliable measure is essential. This makes the quest to establish such a biomarker or biomarkers the highest of priorities.

 

Natural History of HSP
Having a thorough and reliable scientific description of the course of HSP from inception to resolution is of fundamental importance to establishing an effective treatment as it provides the baseline against which measurements are compared. For HSP, with a wide range of genetic causes and huge diversity in the manifestation of symptoms in HSPers, having a good natural history is vital.

 

Regulatory, legal and related requirements
The establishment of an Ethics Committee, or possibly committees, is an early step in the whole process. Their function is to examine plans for the clinical trials and their conduct in detail, and assess their compliance or otherwise with specified ethical considerations.

 

Creating and conducting trials
Trial design
Clinical trials do not come in a ‘one size fits all’ package. Based on the scientific investigation that lies at the heart of a clinical trial, each clinical trial needs to be designed to optimise the potential for a definitive result.

Choice and procurement of drug
Scientific, logistical and financial issues impact on the choice of drug treatment to investigate in the clinical trials. A thorough collation of the relevant data and due consideration needs to be applied to this most important of decisions.

Standardised procedures and processes
Examination, assessment, measurement, recording systems and processes require standardisation across all the medical professionals and sites involved.

Testing
Depending on the choice of drug and biomarker, there will be a need for various forms of testing in the trials. Samples may include blood, urine, cerebrospinal fluid or tissue and may be tested using routine or specialised pathology laboratory services under contract.

Trial implementation
This is the conduct of the clinical trial itself, where participants receive the treatment(s) and various measurements are taken over a period of time. Data is collected, analysed, the results interpreted and conclusions drawn.

 

Participants
Participant recruitment and the processes undertaken for that are complex, with ethical, medical and logistical considerations requiring care in planning and execution. Selection criteria need to be defined and specified. A registry/database needs to be designed and commissioned. Enrolment and consent processes require strict compliance with various standards and protocols. Participant communications to inform and advise represent a significant undertaking in their own right.

 

Organisation
Organisational aspects of clinical trials include management, administration, staffing, planning, accounting including costing and budgeting, IT, communications and reporting functions.

 

Partnerships and funding
A major challenge for a clinical trial such as this is partnerships and funding. Identification of potential pharmaceutical industry partners will be followed by discussion and negotiation to determine if there is sufficient mutual interest to establish a partnership. Grants for conducting clinical trials are offered by organisations such as the National Health and Medical Research Council. Application for such grants is a rigourous and exacting documentation process requiring significant resources and expertise.

Read more …

 

Validating Therapeutic Drug Candidates for treating HSP 2014-16

Validation studies of drug candidates were completed in late 2016. This comprises 2 studies – one with HSP mice and the other with human HSP neurons derived from a different type of stem cell –  induced pluripotent stem cells. Testing of the drug candidates (assays) was identical to the earlier tests performed on the HSP nasal stem cells. Results of the mouse study were inconclusive due to a number of contributing factors, however there were no adverse findings. In the iPSc study, the objective was to successfully differentiate human neurons and again test the drugs on them. This tissue is identical to the nerve cells in the brain where HSP impairment originates and to where drugs will need to be transported in order to be effective. This study validated the earlier results in the nasal stem cells.

Success with the drug candidates in this study supports now taking one of them into clinical trials to test the potential to be an effective treatment for HSP. Here is the September 2016 research progress report.

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Testing & Selecting Therapeutic Drug Candidates for treating HSP 2013-14

This phase of research Testing & Selecting Therapeutic Drug Candidates for treating HSP  was successfully concluded in 2014. With promising candidate drugs having been identified, the research work delved deeper into how HSP affects cell function and the mechanisms, pathways and processes involved in nasal HSP stem cells. Development and refinement of both neuron-like stem cells and the technologies to measure and quantify cell structures and functions will continue in the quest for reliable validation of results.

Two promising candidate drugs are being investigated to determine their suitability for undertaking clinical trials. The current drug investigations are being carried out with nasal HSP stem cells. During this process a 3rd promising candidate drug was identified.

 

Identifying Therapeutic Drug Candidates for treating HSP 2011-12

Results and Reflections

The results of this project were published in January 2013 in the journal Disease Models and Mechanisms.

Prof Alan Mackay-Sim reflected on this two-year project in February 2013 in this article.

Research Project Progress Report, August 2012

Prof Alan Mackay-Sim provided the final progress report before the completion of this two-year project in August 2012.

Research Project Progress Report, May 2011

Principal researcher of the HSP stem cell research project, and Director of the National Centre for Adult Stem Cell Research, Professor Alan Mackay-Sim provided the following progress report on 13 May, 2011. He reports that the project has advanced according to schedule, with the first four aims achieved and the fifth currently progressing. Below is the detailed report:

Identifying therapeutic drug candidates for treating HSP

1.     increase understanding of the consequences of changes caused by mutations in the SPG4 gene, the most common HSP related gene, responsible for 40% of all HSP occurrence.

Everyone has two copies of every gene. These people have one “good copy” and one “bad copy”. We have shown for the first time that patients have a 50% loss of the spastin protein (made from the SPG4 gene). This means that they cannot compensate for the loss of one gene copy by just making more protein from the good copy. This is a new understanding of how the gene mutation works to cause disease. It immediately rules out several hypotheses that people had about the disease. We have shown for the first time that although patients cannot compensate by making more spastin, they compensate by making more of other proteins that work in parallel, with the result that overall, many cell functions are “normal”. This must be true because SPG4 mutations only affect specific nerve cells, in adult life, so the cellular compensations are effective for most cells for all of life and only fail in specific nerve cells in adulthood. Our research now points us to how cells compensate and where they do not.

 

2.     lead to an understanding of the major cellular dysfunctions: detect the dysfunctions/abnormal behavior in cellular pathways; what cell functions are changed/how do cells work in a disease situation.

We have identified for the first time specific cellular pathways that are not fully compensated in patient cells. These new results support existing hypotheses, based on other’s experiments exploring spastin function in non-patient cells. The new results also point to new hypotheses of what is going wrong.

 

3.     define and describe specific cell functions as candidate targets for therapeutic drugs.

We have identified specific changes in microtubule functions inside cells. Microtubules form part of a cell’s “skeleton” at the same time as forming a “roadway” for transporting the specialised “organelles of cell function” around the cell. We have shown for the first time that patient cells have less of one of the proteins that constitute the microtubules. We have identified for the first time two types of organelles whose transport is affected.

 

4.     devise assays for subsequent screening of therapeutic drugs against the target cell functions.

We now have three assays that may be suitable for screening drug libraries: assays for the microtubule proteins and assays for the organelles. Our institute recently purchased the instrument on which the assays were devised when it was on loan from the company. The instrument costs $500K.

 

5.     validate the assays.

We are just starting the validation phase. We have a strategy based on an understanding of the known functions of the spastin protein and on our own experiments, with drugs known to interfere with microtubule function.

 

Research Project Description

The research provider for the HSP Research Foundation (HSPRF), the National Centre for Adult Stem Cell Research(NCASCR) located at Griffith University in Queensland, has just successfully completed a pilot study, the first part of a research program Towards a Cure for HSP to discover and develop drugs for treating HSP.

In the Pilot Study, olfactory stem cells from HSP patients and controls have been successfully differentiated into neurons. Gene expression profiling was done and altered cell functions identified and validated.

This new Project over 2 years is the next step in the program. The aim of the project is to identify therapeutic drug candidates for treating HSP using olfactory (nasal) stem cells from patients as cellular models of HSP.

The first year of research aims to:

  1. Increase understanding of the consequences of changes caused by mutations in the SPG4 gene, the most common HSP related gene, responsible for 40% of all HSP occurrence
  2. Leading to an understanding of the major cellular dysfunctions: detect the dysfunctions/abnormal behavior in cellular pathways; what cell functions are changed/how do cells work in a disease situation?
  3. Define and describe specific cell functions as candidate targets for therapeutic drugs.

The second year of research will aim to:

  1. Devise assays for subsequent screening of therapeutic drugs against the target cell functions.
  2. Validate the assays.
  3. Identify any prospective drugs that emerge through the validation process.
  4. Screen a compound library against targets to identify potential therapeutic drug candidates that compensate for cell functions altered by HSP.

Because the NCASCR research directly targets the disease in human tissue as opposed to lab animals, confidence in positive results for the subsequent stage of drug development is warranted. This project’s outcomes will be the basis of an NH&MRC proposal for the drug development stage.

The 2 year Research Project requires the HSPRF to find $100,000 per year for a post-doctoral scientist at an annual cost of $80,000, plus consumables of $20,000 per year.

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Stem Cell Pilot Study 2009-10

What is it?

The National Centre for Adult Stem Cell Research (NCASCR) and the HSP Research Foundation (HSPRF) have collaborated to implement a Pilot Study aimed at:

  • Growing and maintaining olfactory stem cell lines
  • Differentiating them to other nerve cells and
  • Defining biological differences in SPG4 cell lines from normal cell lines.

This can lead to the definition of drug targets, that is, compounds that may promote normal cell function instead of impaired cell function caused by the particular mutation. Achievement of the study’s goals will lead on to the NCASCR making a submission to the NH&MRC for a grant to then try to identify and validate one or more drug targets.

The Pilot Study is expected to take 12 months and will cost about $100 000, which the HSPRF has raised for the purpose.

Who are the players?

The NCASCR is part of Griffith University’s Eskitis Research Institute for Cell and Molecular Therapies and brings together cell and molecular biologists, structural and chemical biologists and pharmaceutical and medicinal chemists with the goal of discovering new therapies.

The HSPRF was formed in February 2005 at a meeting of HSPers (hereditary spastic paraplegia) at Concord Hospital. An Australian HSP community continues to develop being centered on its website. The HSPRF purpose is to promote research into the detection and prevention of HSP and to advance control of the disease and alleviation of suffering for people with HSP. The researchers include:

Principal Investigator
Professor Alan Mackay-Sim, BA PhD
National Centre for Adult Stem Cell Research
Eskitis Institute for Cell and Molecular Therapies
Griffith University

Co-Principal Investigator
Associate Professor Carolyn Sue, MBBS, PhD, FRACP
Kolling Institute of Medical Research, University of Sydney
Royal North Shore Hospital

Research Scientist
Greger Abrahamsen
National Centre for Adult Stem Cell Research
Eskitis Institute for Cell and Molecular Therapies
Griffith University

The Research

The basic question addressed by the pilot study is:

How do HSP SPG4 gene mutations affect cellular function?

The proposed research targets Hereditary Spastic Paraplegias (HSP), caused by mutations in the SPG4 gene. The overall objective of this pilot study is to identify a set of cellular targets for future therapies for HSP by gaining a better understanding of the cellular consequences of SPG4 gene mutations. The cell model is neural stem cells from patients with SPG4 mutations. The methodological approach is to investigate gene expression in neural stem cell lines from individuals with different mutations in SPG4 and compare this with gene expression in neural stem cells from healthy controls.

The specific aims of this pilot study are:

  1. Identify SPG4 patients and build a clinical data base of patients and controls (CoPI)
  2. Identify specific SPG4 mutations in blood of individuals (genotyping) (CoPI)
  3. Grow neurosphere cultures from patients and controls (CoPI, PI)
  4. Genotype neurosphere-derived cells (PI)
  5. Perform expression profiling of neurosphere-derived cell lines (PI)

The hypotheses to be tested are:

  • Gene expression in olfactory stem cell cultures of SPG4 patients is different from that in cells derived from healthy controls and from patients with other neurological diseases.
  • Gene expression profiling will identify altered gene networks and altered cellular pathways common to patients with SPG4 mutations.

The outcomes at the end of the year-long project will be:

  1. A library of adult neural stem cell lines from SPG4 patients and controls
  2. A database of clinical presentation, blood cell genotype, olfactory cell genotype and gene expression profiles of cell lines from each patient
  3. A database of identified differences in gene expression in patients and controls
  4. A database of gene networks and cell signaling pathways altered in HSP SPG4
  5. A set of cell lines for future studies (eg functional analyses, protein analysis, drug candidate analysis)
  6. A set of potential cellular targets for future therapies.

The timeline for the study will be:

  1. Recruit participants and perform biopsies of olfactory mucosa – 1 to 6 months
  2. Grow patient and normal cells from olfactory stem cells in culture- 1 to 6 months
  3. Differentiate olfactory stem cells to relevant neurons and glia- 1 to 6 months
  4. Identify gene expression and biological pathway differences- 3 to 9 months
  5. Validate gene expression differences and biological pathway difference – within 12 months
  6. Document outcome of study and communicate to funding bodies within 12 months

Facilities

The National Centre for Adult Stem Cell Research is funded by the Australian and Queensland Governments to investigate the biology and clinical application of stem cells derived from adult tissues. The Centre is housed in new $30M laboratories that include facilities for human cell culture, advanced microscopy, cell and molecular biology, and gene expression microarray.

The Centre staff currently comprises 20 PhD scientists, 15 technical staff, and 10 PhD students located at the central labs at Griffith University, Brisbane. An additional node is located at the Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, with 2 PhD scientists, 2 research assistants and 1 PhD student. The senior staff include specialist neurologists, neuroscientists, cell and molecular biologists, bioinformatics and computing specialists.

In cell-based resources, the Centre has an extensive library of adult neural stem cells from healthy controls and people with various neurological conditions. Currently there are cell lines from over 50 people with conditions such as Parkinson’s disease, schizophrenia, motor neuron disease, and mitochondrial disorders. Gene expression profiling is available on over half of these cell lines, which provides valuable comparison data for profiling of SPG4 cells. The Centre has developed databases to house clinical, technical, biological and genetic information on all its cell lines. These are incorporated into a laboratory management system to allow detailed analysis and comparison across all patients and all stem cell lines.

The Centre is part of the Eskitis Institute for Cell and Molecular Therapies. The Institute is home to Australia’s premier facility for high throughput screening for drug discovery. The Institute houses the Queensland Compound Library, Australia’s only compound management and logistics facility, with capacity to store over 200,000 individual compounds and ‘Nature Bank’, a unique collection of over 200,000 optimised natural product fractions derived from over 18,000 samples of plants and marine invertebrates from Australia, China, and Indonesia. These collections are unique resources available for drug screening and the development of drugs against new targets, such as those identified by the current proposal. Read more about how stem cells can be used to lead to drug discovery.

Background and Significance

Hereditary Spastic Paraplegia (HSP) refers to a group of inherited neurological disorders involving progressive weakness and stiffness in the lower leg muscles. The early signs include difficulties with walking, a limp and early falls. Symptoms are progressive, with increasing use of walking aids. Some patients eventually require a wheel chair. It is caused by inheriting of a mutation in one of 32 genes, with about 40% being caused by a mutation in SPG 4.Age of onset varies from very young to over 70 years of age with the peak onset in early adult hood.

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The economic and social impacts of HSP on the community must be considered, not only in terms of direct subsidies for medical treatment, living subsidies and social assistance provided by Governments, social organisations and charities, but also in the costs to the individual and community in terms of lost use of training, experience and loss of employment and productivity. Costs to the individual and family include continued and increasing burden of care, depression, and loss of activities of a healthy work and family life.

Due to the variable age of onset, gene mutation carriers live with the uncertainty regarding their disease status. Carriers often have an active/athletic youth and a walking impaired adulthood. The uncertainty may have a negative impact on the psychological well being of at risk individuals and their families-especially regarding family planning. Genetic counseling services are used and diagnostic gene tests are available for an estimated 70% of all cases. Some patients can successfully delay progression with physical therapy and more advanced cases may require a Baclofen pump to reduce spasms. Some patients have had their mobility enhanced through surgery on tendons. Unfortunately there is no cure.

A promising pathway towards a cure

A fuller understanding of the cellular basis of the disease may lead to therapies that target and ameliorate the effects of the genetic mutation. With the potential availability of genetic screening it may be possible to identify individuals at risk for HSP and treat them prophylactically.

The protein for which SPG4 encodes, spastin, is a microtubule-severing protein, thought to be important for intracellular transport along microtubules (Evans et al., 2006). Mutations in SPG4 are thought to interfere with intracellular trafficking, the consequences of which would be logically more severe in central and peripheral motor neurons whose axons are the largest in the body. The consequences of gene mutations are often studied in simple cellular systems (yeast, fungi, HeLa or Cos cells)(Charvin et al., 2003), simple organisms (Drosophila)(Trotta et al., 2004), or transgenic mouse.

These are all valid and useful approaches but they lack the complexity of the genetic background found in individual patients. This may be important particularly for understanding the consequences of SPG4 mutations because there is no common mutation, rather a variety that occur independently in different families, including missense, nonsense, frameshift, and splice site mutations, and deletions.

The cellular role of spastin is not understood, nor are the consequences of SPG4 mutation. The relationship between cellular function and clinical phenotype is also unknown (eg age of onset, complications additional to pure HSP). Some mutations suggest a “dominant-negative” cellular mechanism in which the mutated protein blocks or alters the function of proteins with which spastin might normally interact, although this is disputed (Schickel et al., 2006). Without comparison of different cells, this is hard to reconcile with deletion mutations in which the spastin protein is lacking entirely.

This proposal presents a strategy to develop stem cell lines from multiple individuals with SPG4 mutations which can then be compared to investigate the cellular mechanisms altered by SPG4 mutations allowing identification of cellular pathways common to and differently altered by different mutations and those specific to individual mutations.

Olfactory stem cells as cellular models of neurological disease

Stem cells in the adult olfactory mucosa normally provide the capacity for sensory neuron replacement that occurs throughout adult life (Mackay-Sim and Kittel, 1991; Leung et al., 2007). The olfactory stem cell is multipotent with the ability to generate neuronal and non-neuronal cell types in vitro and after transplantation into the developing embryo (Murrell et al., 2005). Olfactory stem cells can be grown in vitro as neurosphere cultures for many generations without altering their phenotype (Roisen et al., 2001; Murrell et al., 2005).

Olfactory stem cells are neural stem cells available in all adults. As stem cells they can be grown indefinitely in culture, frozen, stored and revived. They can also be differentiated into many cell types, including the neurons and glia of the nervous system. This provides the opportunity to use olfactory stem cells as “cellular models” of disease. An analogy is the use of breast cancer cell lines as cellular models of breast cancer and which distinguish between different types of breast cancer to develop new diagnostics and treatments. In our laboratory we are building up a library of olfactory stem cell lines from healthy controls and from patients with different neurological conditions. We currently have over 50 cell lines from people with Parkinson’s disease, motor neuron disease, schizophrenia, and mitochondrial disorders.

Gene expression profiling using microarray

Gene expression microarray profiling is a technique that identifies which genes are active in a cell. The principle is to identify the mRNA that is expressed. mRNA is transcribed from the DNA and is translated by the cellular machinery into proteins. Identifying the mRNA expressed in a cell gives an idea of the proteins the cell is making. By using microarray technologies that utilise the whole human genome, one can identify networks of genes/proteins that are active and inactive in cells. This provides insights into the cellular pathways that are important in different cell types or in different disease states.

Gene expression in olfactory stem cells in neurological diseases

To date we have investigated gene expression in 44 human olfactory stem cell lines using the Ilumina Beadstation microarray platform. These include cells from healthy controls, patients with schizophrenia and patients with Parkinson’s disease. This gene expression platform gives highly reproducible data indicated by a very high correlation between gene expression in two replicates of independent cell lines generated from the same person (r=0.99, Fig 1).

Fig.1Gene expression in biological replicates is highly correlated. Raw fluorescence values from microarrays probed with mRNA from cell line1 (y axis) correlated with mRNA from cell line 2(x axis) from same patient R=0.99

Fig 2. Gene expression in olfactory stem cell culturesdistinguishes patients with schizophrenia (red columns) from healthy controls (blue columns). Rows represent genes with increased expression (red bars) or decreased expression (green bars).

This method has identified 1053 genes whose expression is altered in schizophrenia (n=10, Fig2) and 2024 genes altered in Parkinsons disease (n=20). Using these set of genes we have identified several cellular pathways that are altered exclusively in these diseases. In schizophrenia it is obvious that cell cycle genes and neural differentiation genes are altered (McCurdy et al., 2005). It should be noted that gene expression in fibroblasts is not different in schizophrenia versus controls. Fibroblasts provide a comparison, non-neural cell line, to test the specificity of the olfactory stem cell lines as models for neurological disease.

These experiments indicate that olfactory stem cells provide a useful model in which to investigate cellular functions that are altered in neurological diseases.

Expression of HSP genes in olfactory stem cell cultures

About 30 genes have now been identified that lead to HSP, some of them associated with additional syndromes (Zuchner, 2007). It is a challenge to imagine a unified cellular explanation for all presentations. The present proposal centres on SPG4/spastin. Preliminary analysis of the microarray data obtained from 88 olfactory cell lines (from 44 individuals) indicates that SPG4 and other HSP genes involved in intracellular trafficking (SPG3A, SPG7, SPG20, SPG21) are expressed by all cell lines. Additionally, all cell lines express two HSP genes involved in mitochondrial function (SPG7, SPG20). These data suggest that these cell lines will be useful for studying the role of SPG4 mutations, including the consequences on other genes of interest in HSP.

Research Design

The overall design is to use adult neural stem cells obtained from SPG4 patients as cellular models of the disease. Adult neural stem cells are obtained from the olfactory mucosa, the organ of smell, via nasal biopsy, from SPG4 patients and healthy controls. These olfactory stem cells are grown in vitro and SPG4 -Control differences in gene expression will be used to identify cellular targets for future therapies.

The rationale is that olfactory stem cells are multipotent and can give rise to neurons and glia, the cells of the nervous system. The overarching hypothesis is that any cellular effects of genetic mutation in neurological diseases will be manifest in neural stem cells cultures and the cell derived from them in vitro.

The proof-of-principle of this approach has been obtained in studies of olfactory stem cells inpatients with Parkinson’s disease and in schizophrenia (see above). Bioinformatic analysis of the disrupted gene expression identifies networks of genes and distinct cellular signaling pathways that are

altered in each of these neurological conditions. To date, our gene expression analyses have been confined to early stem/progenitor cultures, but even these contain differentiating neurons. These data indicate significant expression in all cell lines of neuronal genes such as neurotransmitter receptor genes, synaptic vesicle genes, axon guidance genes and neural growth factor receptor genes. Of interest here are the high proportion of genes expressed that are associated with neurodegenerative conditions such as Huntington’s disease, Alzheimers disease and Parkinson’s disease. To investigate the cellular pathways and gene networks altered in SPG4, gene expression will be analysed at different stages of differentiation from the early stem/progenitor cultures through to cultures of differentiated neurons. Olfactory stem cell culture assays demonstrate significant differences in cellular functions in schizophrenia and Parkinson’s disease. We also have data from primary cell culture experiments demonstrating functional differences in schizophrenia (McCurdy et al., 2005) and in olfactory cell lines from patients with mitochondrial gene mutations (Sue et al, unpublished).It should be noted that the patients in our current studies do not have a known gene mutation – the differences in gene expression and cellular function are probably due to multiple gene mutations of small effect combining similarly in each of the patient groups. In the case of SPG4, with defined, clinically manifest mutations, there can be no doubt that the technique is sensitive enough to identify genes and pathways significantly altered by the mutated gene common to all patients and manifest in all the patients’ cells but not in healthy controls. Our database of gene expression in the other diseases provides further discrimination.

The specific hypotheses are:

  1. That cells derived from olfactory stem cells from patients with SPG4 mutations will have altered gene expression compared to genes from healthy controls and from patients with other neurological disorders and diseases; and
  2. That gene expression analyses will identify disease-specific genes, gene networks and cellular signaling pathways. An example of this approach is seen in Fig. 3 which illustrates multiple genes in a cellular pathway whose expression is affected in schizophrenia.

Methods

Olfactory neural stem cells are obtained from nasal biopsies (Feron et al., 1998), prepared according to our published protocols (Murrell et al., 2005). Neural stem cells grow in “neurospheres”, mixed cultures of stem cells, neuronal progenitors and developing neurons and glia. In brief, olfactory biopsies are obtained via the external naris. The cells of the olfactory mucosa are obtained by eznymatic digestion and mechanical disruption of the olfactory mucosa. Primary cultures of these cells are grown for a week in a medium containing 10% fetal calf serum (DMEM/10%FCS). Cells are then dissociated with trypsin and passaged onto plates coated with poly-L-lysine and grown in a neurosphere-forming medium, serum-free medium containing EGF and FGF2 (DMEM/EGF/FGF2).

These cultures form neurospheres after 2 days which form on the culture well surface and break away into the medium. Neurospheres are heterogeneous, containing neural stem cells, progenitors, developing neurons and developing glia. The neurospheres are harvested, dissociated and grown on tissue culture plastic in DMEM/FCS for baseline gene expression studies. Neurosphere-derived cells can also be differentiated into more mature cell types, neurons, astrocytes and oligodendrocytes for further gene expression and cell biological studies.

Gene expression profiling of mRNA is obtained using the Illumina Bead Station which compares the level of gene expression across 50,000 mRNA transcripts representing all the known genes in the human genome. Statistical analysis is used to reveal specific genes whose expression differs between patients and controls. Bioinformatic analysis reveals networks of genes that are coregulated in patient and control samples. It also reveals cell signaling pathways whose genes are affected by the disease.

 

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