Motor neurons are the nerve cells in the body responsible for controlling movement.  A number of diseases are caused by damage to motor neurons, including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).  In order to treat these diseases, scientists are developing methods to generate new, healthy motor neurons from stem cells. A recent study has elucidated the cellular mechanisms that control the motor neuron differentiation, paving the way for new treatments for motor neuron diseases.

Each time we voluntarily move an arm or leg, or when our lungs involuntarily expand and contract, signals from the brain are sent along a chain to the spinal cord, where motor neuron cell bodies reside. These motor neurons terminate in muscle cells, where they transmit the nerve impulses in order to produce muscle contractions. In ALS, there is a progressive destruction of motor neurons due to either a genetic defect or an unknown environmental trigger. Motor neuron damage in ALS leads to progressive muscle weakness that affects all parts of the body, impairing the ability to speak, swallow, and eventually breathe. SMA is caused by gene mutations and is characterized by similarly progressive damage to motor neurons that causes muscle weakness. If respiratory muscles are affected, SMA can be fatal.

Scientists aim to develop gene therapies for these diseases that can repair the damaged motor neurons and improve the functioning and lifespan of patients. To do this, they must first understand the signals that induce motor neuron development from stem cells. Stem cells are the precursors for every type of cell in the body. They are triggered to differentiate into various cell types via cellular signaling molecules called transcription factors, which act on DNA to turn on specific genes. Which genes are turned on will determine the phenotypic fate of each cell. Typically, each cell goes through several stages of development before reaching its final fate.

A group of researchers from several universities recently teamed up to elucidate these programming pathways. They had previously discovered that a group of transcription factors called the NIL factors – Ngn2, Isl1, and Lhx3 – can induce motor neuron development from embryonic stem cells without passing through any of the intermediate stages. Moreover, the NIL factors achieved the transition to the motor neuron fate with a 90% success rate, and the process took only two days. This so-called direct programming pathway was an exciting finding with respect to clinical applications, because it can be achieved both in vitro and in living organisms at the site of cell damage.

In the current study published in the journal Cell Stem Cell, Esteban Mazzoni and colleagues further investigated the process by which transcription factors bind to and activate parts of DNA during the first 48 hours after NIL expression. First, the researchers used single-cell RNA sequencing (RNA-seq) to study the timing of gene expression after induction by NIL programming factors. RNA-seq is a technique that reveals the presence and quantity of RNA in a sample at a specific point in time. Thus, as transcription factors turn genes on, these genes are transcribed into RNA that can be measured and quantified.

The researchers also studied chromatin remodeling during motor neuron programming. Chromatin is a tightly-packed form of DNA which regulates the expression of genes through changes in its structure.  Promoters are regions of the DNA where transcription factors bind in order to initiate gene transcription.  Chromatin must undergo structural changes, called remodeling, in order for the DNA to be accessible to transcription factors. Typically, as cells move through the differentiation process, chromatin changes that occur at promoter regions will restrict the differentiation potential of the cell.

To study this chromatin remodeling process, a ChIP-seq time series was performed.  ChIP-seq combines chromatin immunoprecipitation with DNA sequencing to identify the binding sites of proteins that associate with DNA.  Antibodies against the bound proteins are used to extract protein-DNA complexes, and the DNA binding sites can be sequenced. In addition, the researchers used an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) to study chromatin accessibility.  Proteins called transposons incorporate into exposed, or accessible, portions of chromatin. Therefore, identifying the locations of transposons in the DNA can indicate what parts of the DNA are being actively transcribed, or turned on.

This series of experiments revealed information about how genes are turned on and off over the 48-hour process of motor neuron formation.  Initially, the transcription factors Ngn2 and Isl1/Lhx3 induce different sets of genes in parallel. Whereas Ngn2 controls genes associated with generic neuronal differentiation, Isl1 and Lhx3  activate genes specific for spinal cord and motor neurons. As programming progresses, Ngn2 induces the expression of two other transcription factors, Ebf and Onecut. These transcription factors modify the chromatin state to enable Isl1/Lhx3 binding to previously inaccessible sites on the DNA that contain the terminal motor neuron genes necessary to complete the programming process.

These experiments showed that the activities of Ngn2 and Isl1/Lhx3 act in tandem to induce direct motor neuron programming from stem cells. The researchers hope to apply these findings clinically. By triggering this programming pathway in the body, cells in the spinal cord can be induced to differentiate into motor neurons, replacing the neurons that are damaged in diseases such as ALS.

References

Czarzasta J., Habich A., Siwek T., Czaplinski A., Maksymowicz W., Wojtikiewicz J. (2017) Stem cells for ALS: an overview of possible therapeutic approaches. Int J Dev Neurosci. DOI: 10.1016/j.ijdevneu.2017.01.003

Farrar M., Park S., Vucic S., Carey K., Turner B., Gillingwater T., Swoboda K., Kiernan M. (2016) Emerging therapies and challenges in Spinal Muscular Atrophy. Ann Neurol. DOI: 10.1002/ana.24864

Mazzoni, E.O., Mahony, S., Closser, M., Morrison, C.A., Nedelec, S., Williams, D.J., An, D., Gifford, D.K., and Wichterle, H. (2013). Synergistic binding of tran- scription factors to cell-specific enhancers programs motor neuron identity. Nat. Neurosci. 16:1219–1227. DOI:10.1038/nn.3467

Velasco S., Ibrahim M., Kakumanu A., Garipler G., Aydin B., Al-Sayegh M., Hirsekorn A., Abdul-Rahman F., Satija R., Ohler U., Mahony S., Mazzoni, E. (2016) A Multi-step Transcriptional and Chromatin State Cascade Underlies Motor Neuron Programming from Embryonic Stem Cells. Cell Stem Cell. DOI: 10.1016/j.stem.2016.11.006

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