Characterization of spinal motor neuron loss in a mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a neurodegenerative disease most commonly caused by mutation of the Survival Motor Neuron 1(SMN) gene. It is the most common genetic cause of infant death and patients with the most severe form of the disease (type 1) generally do not live past one year. The hallmarks of the disease are muscle weakness and atrophy. The disease is thought to be caused by abnormalities of motor neurons (MNs) and some previous studies from human spinal cord autopsies suggest that there is a loss of large anterior horn cells, presumed to be motor neurons. Advances in transgenic technology have enabled investigators to study this disease in a mouse model. The SMA-Δ7 mouse has a severe phenotype leading to death at the end of the second postnatal week. This model resembles type 1 SMA in humans. We have used this model to investigate the extent of motor neuron death in the lumbar cord of the SMA mouse at postnatal day 4.

In my project, individual spinal motor neurons in mutant (SMA-Δ7) and wild type mice were labeled with two independent methods (see below) to count them and to measure their size. I focused on the motor neurons of the first lumbar segment at 4 days of age for both mutant and wild type mice. Motor neurons were first labeled with a technique developed in Dr. Michael O'Donovan's lab. With the help of Dr. George Mentis, I isolated spinal cords from mice and kept them artificially alive by bathing them in artificial cerebrospinal fluid. With the aid of special electrodes, we labeled motor axons exiting the spinal cord with a fluorescent dye (Texas Red Dextran) which retrogradely fills the motor neuron cell bodies by simple diffusion. After allowing approximately 24 hours for labeling, the spinal cords were fixed in 4% paraformaldehyde. The following day, the cords were cut in thin sections (75 μm) using a Vibratome. The sections were collected in wells and were then processed for immunocytochemistry. I used a primary antibody against choline acetyl transferase (ChAT) which is a well known marker for cholinergic motor neurons. The ChAT antibody was visualized by the subsequent use of a secondary antibody conjugated to a fluorochrome (FITC or Cy5), which was different from the Texas Red Dextran used to retrogradely label the motoneurons. After the immunocytochemistry, I collected the sections and mounted them on slides for inspection under the confocal microscope. I used an LSM510 (Carl Zeiss) microscope to excite the two different fluorochromes and subsequently acquired images. With the confocal microscope I was able to acquire images of small optical thickness (range: 1.5-2.5μm) throughout the whole 75μm section. In this way, I was able to visualize and count all motor neurons that were labeled with both fluorochromes. It was important to use two independent methods to identify motoneurons because it was possible that the disease process in SMA might lead to the down regulation of ChAT which would have caused us to underestimate the number of motoneurons in the SMA mice.

I found that in SMA mice, there is approximately 50% reduction in the number of ChAT+ motor neurons (3 spinal cords from SMA-Δ7 and 3 spinal cords from wild type mice). There were on average 382 13 ChAT+ MNs in wild type and 189 27 ChAT+ MNs in SMA mice in the first lumbar segment (49.5% MN loss; p<0.003, t-test). The number of retrograde filled motor neurons was lower in both types of mice as compared with the ChAT staining. This could occur because some motor neuron axons did not exit the spinal cord or alternatively because not all axons were labeled by the retrograde method. We calculated that approximately 60-70% of motor neurons that are ChAT immunoreactive were labeled retrogradely. Nevertheless, the number of retrogradely labeled motoneurons was also reduced in the SMA mice (wild type retrograde filled MNs: 232 44; SMA: 129 12; 55.6% MN loss; p=0.09, t-test). Finally, I measured the transverse somatic area of all motoneurons (number of MNs: 1146 wild type - 567 SMA). The distribution of the MN somatic area revealed that there is no preferential loss of large (presumed to be α-type) or small (γ-type) motor neurons in SMA mice. On average there was a small reduction of the soma size (wild type: 314.5μm²; SMA: 291.2μm²) in the SMA motoneurons although this did not reach statistical significance.

My results are the first detailed documentation of the extent of spinal motor neurons loss in the SMA-Δ7 mouse model of Spinal Muscular Atrophy. They will help in the understanding of the progression of the disease and my hope is that it will aid towards a potential treatment.