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Identification and Characterization of Factors Critical in Regulating the Selective Vulnerability of Distinct Motor neuron Pools in Spinal Muscular Atrophy Models of Mice

This research project is being conducted at the Ottawa Hospital Research Institute by Dr. Rashmi Kothary and Dr. Lyndsay Murray. This project is on its second year of a 2 year agreement with a total funding agreement of $120,000.00. An interesting report from the researchers follows:

We are currently in our second year of funding from FSMA Canada.  Our research is directed at understanding how motor neurons degenerate in SMA with the hope that we can find ways to protect them.

Motor neurons are exceptionally large cells, extending from the spinal cord to the muscle. The cell body, which is the control centre of the cell, is situated in the spinal cord, while the extremity of the cell is situated within the muscle. In some cases this means that one cell can be over 1 meter long. This remarkable size is important to remember when considering motor neuron pathology, as it is thought that these different parts of the cell degenerate at different times.  There is now considerable evidence that the part of the cell that forms the connection with the muscle, the so called neuromuscular junction, degenerates before the rest of the cell.  As a consequence of this, we believe that if we can protect the neuromuscular junction, then we may be able to protect the whole cell. 

In addition, not all motor neurons appear to be equally vulnerable in SMA.  For example, those motor neurons that supply muscles in the head and neck appear to be less vulnerable than those that supply muscles in the lower limbs.  This observation strongly implies that there can be important differences between different motor neurons populations.  This is very interesting to us for a number of reasons. Firstly, comparing those neurons that are vulnerable to those that are less vulnerable allows us to ask what changes within the cell cause it to be vulnerable.  This can help us understand the reasons why the cell is dying.  Secondly, comparison of these different motor neurons can reveal features that can protect one neuron over another.  If we can identify the factors that are protecting a particular type of motor neuron, then we can use this information to develop therapeutics to help protect the more vulnerable motor neurons.    

To investigate the reasons for these differences in vulnerability, we use a mouse model of SMA, known as the Smn2B/- model.  In this mouse, neuromuscular junctions in the muscles from the neck are preserved whilst those in abdominal muscles degenerate, as shown in Figure 1.  It is our aim to understand what the differences are between these two different populations of motor neurons. In order to do this, we identify the neurons in the spinal cord and brainstem, which connect to either the neck or abdominal muscles. We then dissect them and compare gene activity between groups of motor neurons that are either vulnerable or less vulnerable in SMA.

There are a number of steps involved in these experiments. Firstly, in order to identify which motor neuron cell bodies correspond to the muscles we are interested in, we inject a dye into the muscle. This dye travels up the neuron and accumulates in the cell body (Figure 2). These dyes are fluorescent so when we look under a microscope we can see which cells are labeled. We isolate the motor neurons using lasers to microdissect out individual cells (Figure 3). We then use screening methods and powerful software to compare the gene activity from cells that are vulnerable to SMA and those which are not. 

During year one of this project, we optimized the procedures for this protocol to make sure we could identify cells confidently and to make sure the cells we obtained were of sufficient quality to give informative results.  During year two of this project, we have dissected these cells and gathered enough material for the screening procedures.  This screening is currently underway and we expect to have the results within the coming weeks.  The next stage will be to use software to compare gene activity.  This phase of the project will aim to address two main questions.  Firstly, what makes different motor neurons different from each other?  Secondly, what are some of the first changes that happen at the cell body when a neuromuscular junction starts to degenerate?  By understanding this process, we hope to develop therapeutics that can slow or prevent the degenerative process.

Figure 1: Variability in neuromuscular junction vulnerability in muscles from the abdomen or neck in SMA mice.
This figure shows neuromuscular junctions with either high or low vulnerability.  The red staining represents the part of the neuromuscular junction formed by the muscle.  The green represents the part of the neuromuscular junction formed by the neuron.  In the examples from the healthy mouse, the green staining very tightly correlates with the red staining implying neuromuscular junctions are intact and functional. In theneck muscles from the SMA mouse, again, neuromuscular junctions appear intact i.e. all the red staining is accompanied by green staining.  In the abdominal muscles, there are frequent examples of red staining which has no green staining.  This implies that the neuron has been lost and the neuromuscular junction has therefore degenerated.  Our work aims to ask why we see these different levels of degeneration within an individual mouse. (Click the image to view a large version)

Figure 2: Diagram showing methods employed to identify motor neurons in the spinal cord.
This figure demonstrates the procedures we use to identify which motor neurons correspond to the muscles that we are interested in.  A red fluorescent dye is injected into the muscle of interest and travels along the motor neuron until it reaches the cell body located in the spinal cord.  After euthanizing the mouse, we remove the spinal cord and can identify the motor neurons that we are interested in by looking for the red fluorescence.  An example image can be seen on the right.  Note that all the motor neurons are marked with a green stain, but only 2 of the 3 also display a red stain.  We use this method to identify which cells to dissect out to analyze gene activity. (Click the image to view a large version)


Figure 3:  Example of laser capture microdissection.
This figure demonstrates the process we use to isolate motor neurons.  The spinal cord is cut into sections that are 10 microns thick.  A laser is then fired at the cells we want to dissect.  This allows us to cut out only the cells we are interested in and leave the rest of the tissue intact. (Click the image to view a large version)

Posted May 6, 2013