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Organotypic cultures as tools for functional screening of drugs, full application

To identify molecules activating lineage specific transcription factors to promote Oligodenrodrocyte differentiation and develop automated software to quantify cell number and state.

The Idea

A major challenge for the pharmaceutical industry is the development of relevant model systems in which knowledge gained from high-throughput, genomic and proteomic approaches can be integrated to study function. The level of complexity of high-content systems is steadily advancing and in vitro functional bioassays based on organ systems are rapidly emerging as an important new interface technology between discovery and development. Animal models are still the main choice for such studies but over the past few years powerful new in vitro systems have begun to emerge as useful tools to study function. Organotypic cultures made from slices of explanted brain tissue represent a complex multi-cellular in vitro environment with the potential to assess biological function and are uniquely placed to act as an important link between high-throughput approaches and animal models.

Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. In response to myelin degeneration multipotent parenchymal progenitor cells called oligodendrocyte progenitor cells (OPCs) are activated and recruited to the damaged areas. These can regenerate myelin to some extent, but the process is often incomplete, leaving axons permanently demyelinated and vulnerable to degeneration. These unrepaired areas causes the symptoms seen in patients with demyelinating diseases, including blurred double vision, clumsiness, hemiparesis, paresthesias, impaired muscle coordination, loss of sensation, unsteady gait, and hearing and speech problems.

Remyelination of lesions often remains incomplete despite the presence of OPCs, particularly in MS. The reasons for remyelination failure in MS are still not fully understood, and are expected to be complex. However, it has been demonstrated that OPCs that fail to differentiate into remyelinating oligodendrocytes are present in a proportion of demyelinated lesions. Failure of OPC differentiation is a major cause of remyelination failure. Currently there are no pharmacological therapies exist that promote OPC differentiation and regeneration capacity of the demyelinated human CNS.

In this proposal we aim to identify molecules that will be identified by Cheminformatics approach to activate lineage specific transcription factor as to promote Oligodenrodrocyte differentiation. Secondly, the identified molecules will then be screened on brain slice culture to test it efficacy to promote oligodendrocyte differentiation and myelination. Thirdly, we will build up automated software to quantify the level of mature oligodendrocytes and myelination.

Who we are

Yasir Ahmed Syed - CNS regeneration

Patrick Rericha - Oligodendrocytes

Mark Kotter - CNS remyelination

Andreas Bender - Medicinal Chemistry

Implementation

Organotypic brain slice cultures occupy a unique niche in the drug discovery process and are a unique tool in the lead discovery and validation process. while animal models are the most biologically relevant systems, they represent the most difficult system in which to study new targets and new leads. Testing new compounds in animal models is limited by the need for skilled personnel. In many cases they are also very time-consuming. For example mouse models of Alzheimer’s can take several months to develop a disease phenotype. They are also expensive and increasingly unpopular on ethical grounds. High-throughput screening is easiest to perform in cellfree systems; however, the likelihood of discovering molecules that act on novel targets is extremely low since one can only detect hits that interact with this isolated target or target system. Clearly what is needed now is the development of intermediary cell-based systems that have sufficient throughput but that encapsulate enough biological complexity to enable pre-selection of hits for further in vivo studies. The closer in vitro models parallel in vivo models and reflect pathophysiological mechanisms related to human disease conditions, the more likely they are to be of value as interfaces between drug discovery and development. The level of complexity of high-content systems is steadily advancing and in vitro functional bioassays based on organ systems are rapidly emerging as an important new interface technology between discovery and development. These are referred to generally as organotypic models and have traditionally relied on acutely isolated tissue preparations such as liver or brain slices, which can be maintained for several hours to weeks ex-vivo.

The quest for discovery of molecules that improve CNS remyelination is prompted by the premise that this will not only help repair demyelinated lesions in multiple sclerosis (MS), aiding symptoms by overcoming conduction block and temporal dispersion of action potentials by restoring saltatory conduction, but also protect axons from neurodegeneration which causes progressive disability. Myelinating oligodendrocytes have been shown to be trophic for axons, providing them with necessary metabolic support. Remyelination does occur in MS brain, but is inefficient and inadequate. For remyelination to occur, oligodendrocyte precursor cells (OPCs) must survive, proliferate, migrate to the lesion and differentiate into mature oligodendrocytes forming compact myelin sheaths. Failure of remyelination may occur at any step in this process, but it is thought to be mostly due to failure or arrest of oligodendroglial differentiation. This is partly due to pathological studies revealing that 60–70 % of demyelinated MS lesions contain oligodendroglial cells in an arrested maturation state. Hence promoting these arrested oligodendroglial cells to differentiate present a therapeutic avenue to treat demyelinating disease.

My research in lab of Mark kotter has been to identify molecules that promote or inhibit oligodendrocyte differentiation. I have identified EphrinB3 and Semaphorin3a as inhibitors of Oligodendrocyte differentiation and CNS remyelination. Recently, I have developed organotypic brain slice culture to study the dynamics of Oliogendrocytes along with my student Patrick Rericha.

Aims

1) To identify the small molecules that activate OPC specific lineage transcription factor.

In collaboration with Andreas Bender (medicinal chemistry), I aim to identify the small molecules that target Oligodendrocyte transcription factor and myelin genes as to drive oligodendrocyte differentiation and CNS myelination using chemiformatics based approach. We will used the microarray data to identify genes that are specifically regulated during early and late differentiation phase. Following the identification of genes, we will identify a small molecule that activates the lineage specific factors using the software that has been developed in Bender lab.

2) Functional screening of identified small molecules on brain slice culture

The identified molecules with good score after target validation would be added in medium used to culture the brain slices. Mice Brain slices of 300µm thick would generated using manual tissue chopper. Appropriate positive and negative control would be used to evaluate the efficacy of identified molecules. The slices would be cultured for 10 days, following which the slices would be fixed and stained for mature markers of oligodendrocyte differentiation (MBP, CNP). Following which it will be imaged using confocal microscope. My student Patrick Rericha will assist me in performing the experiment.

3) To develop automated software as to quantify differentiated oligodendrocytes and myelination.

To date the quantification of OPC differentiation and myelination is done manually with investigator blind folded. Often this leads to error, as some time neighboring cells are not easily distinguishable because intermingled myelin sheath. To have an unbiased approach, developing quantifiable software that distinguishes cells, its morphology and to quantify the intensity of staining is the need of hour. Together with Andreas Bender, I aim to develop the software that would aid in quantification of differentiated cells not only on slice culture, but also in vitro cultured cells. The software would be designed as such to use it as plugins for ImageJ and Cell profiler.

Benefits and outcomes

The proposed aims fits well with objectives of SRI as the generated result would yield tangible outcome that can be readily shared across scientific community in span of six months. The proposed aims fits well with focus areas of the SRI and will result in open technology and innovative platform for drug screen. The results gained would be of immense importance to the field of CNS remyelination as currently no therapies exist to treat disabling demyelinating disease like multiple sclerosis.

The proposed organotypic slice cultures have the potential to become powerful tools in the arsenal of drug discovery technology, lying at the interface between high-throughput screening and clinically relevant pre clinical animal disease models. I believe that they can act as a useful starting point for refining molecules for further in vivo analysis and reduce the number of potential leads one needs to evaluate in animals. In addition they can be used to functionally screen compounds in the absence of target information. The data would be made public by publishing it in open access journals.

The software that would be generated to automate the quantification of closely packed membranous oligodendrocyte would be made publically available. People would shared interest could use it and modify it for their intended application. Most importantly, the proposed project is interdisciplinary (Cell Biology, medicinal chemistry, cheminformatics, tissue engineering) and would aid in fostering the collaboration with Andreas Bender lab.

Budget

  • Consumables - £3000
  • Microarray - £800
  • Animals purchase - £800
  • Softwares - £400

My research has been funded by UK MS society. Any additional cost required for publication, travel would be covered by the fund and research supervisor has agreed to it.

About

The Synthetic Biology Strategic Research Initiative provides a hub for anyone interested in Synthetic Biology at the University of Cambridge, including researchers, commercial partners and external collaborators. 

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