Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis - vulnerability of lower motor neurons to proximal excitotoxicity

Catherine A Blizzard, Katherine A Southam, Edgar Dawkins, Katherine E Lewis, Anna E King, Jayden A Clark, Tracey C Dickson, Catherine A Blizzard, Katherine A Southam, Edgar Dawkins, Katherine E Lewis, Anna E King, Jayden A Clark, Tracey C Dickson

Abstract

There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.

Keywords: Amyotrophic lateral sclerosis; Excitotoxicity; Excitotoxin exposure; Lower motor neuron; Motor neuron disease.

Figures

Fig. 1.
Fig. 1.
In vitro model of site-specific excitotoxicity. (A) Primary spinal motor neurons were grown in microfluidic chambers. (A) SEM analyses demonstrated primary motor neuron cell bodies (left) spatially separated from C2C12 muscle cells (right) in the distal chamber with interconnecting axons. (B–G) Immunocytochemistry for the cytoskeletal markers that are enriched in dendrites (MAP2, red) and in axons (SMI32, green). (B) Proximal chamber with vehicle control has neurons and continuous neurites immune-positive for both MAP2 (red) and SMI32 (green). (C) The proximal compartment, following treatment of the distal compartment with kainic acid, has neurons and continuous neurites positive for both MAP2 (red) and SMI-32 (green). (D) The proximal chamber, following treatment with kanic acid at the proximal end, demonstrated a loss of immunoreactivity for both MAP2 (red) and SMI32 (green). (E) The distal compartment following treatment with the vehicle control has continuous neurites that are immuno-positive for SMI32 (green). (F) The distal compartment following treatment with kainic acid at the distal end has continuous neurites that are immuno-positive for SMI32 (green). (G) The distal compartment, following treatment with kainic acid at the proximal end, demonstrated axonal degeneration (green). (H) Quantitation of axon degeneration in the distal chamber after treatment with vehicle control, or application of kainic acid (KA) to the distal or proximal compartments. A significant increase in total distal axon degeneration following treatment with kainic acid at the proximal end was seen. Furthermore, there was a significant (*P<0.05) increase in axon fragmentation in the distal compartment following exposure of the proximal compartment to kainic acid. Error bars represent s.e.m. Scale bars 100 μm (A); 50 μm (B–G).
Fig. 2.
Fig. 2.
In vivo chronic exposure of the spinal cord to kainic acid – proximal effects. (A) Osmotic mini pumps were placed subcutaneously with the catheter inserted into the subarachnoid space at L5 of YFP-expressing mice. (B) Fluoro-Ruby (red) labelling was present at L4 of the spinal cord of YFP-expressing transgenic mice (green) after 7 days of infusion. (C–F) Fluoro-Ruby (C, red), YFP (D, green) with immunohistochemistry for SMI32 (E, blue) and merged (F), in the anterior ventral horn of the L4 region, demonstrated that kainic acid was being taken up by motor neurons of the spinal cord. (G,H) Toluidine Blue staining following treatment with the vehicle in the anterior ventral horn (H, inset of G) of the spinal cord at L4 on day 28 of the infusion time course. (I) Toluidine Blue staining in the ventral horn of mice treated with an infusion of 10 mM KA for 28 days (arrowheads) demonstrated cell loss. (J) Confocal microscopy demonstrated that there was axonal fragmentation (arrowheads) in the dorsal corticospinal tract after 28 days of infusion. (K) Quantitation of Toluidine-Blue-stained motor neurons in the anterior ventral horn of the spinal cord in the L3 to L4 region after 7, 14 and 28 days of infusion (INF) demonstrated that there were no changes between animals that had been treated with vehicle control and 10 mM kainic acid (KA) for 7 days. There was a significant (*P<0.05) reduction in the mean number of motor neurons at days 14 and 28 in the spinal cords that had been treated with 10 mM kainic acid in comparison with those treated with vehicle control. There was also a significant difference between animals treated with 5 mM and 10 mM kainic acid after 14 and 28 days of infusion. Error bars represent s.e.m. Scale bars: 120 μm (B); 40 μm (C–F); 100 μm (G); 50 μm (H,I); 100 μm (J).
Fig. 3.
Fig. 3.
In vivo chronic exposure of the spinal cord to kainic acid – distal effects over a live-imaging time course. (A) Confocal microscopy images of YFP-labelled NMJs in the gastrocnemius muscle. (B–D) NMJ trees of vehicle-control mice were relatively stable (arrowheads) over the imaging time course at days −14 (B), 14 (C) and 28 (D). (E–G) An NMJ tree of mice treated with 10 mM kainic acid over the imaging time course at days −14 (E), 14 (F) and 28 (G) underwent morphological alterations (F; arrowheads) and NMJ loss (G; arrows). (H) There was a significant increase in the number of NMJ branch points at days 14 and 28 for the kainic acid (KA)-treated mice. (I) Quantitation of the mean NMJ area demonstrated a significant (*P<0.05) reduction in the mean NMJ area at both day 14 and day 28 for the KA-treated mice compared with vehicle control. There was no significant difference between the results at days 14 and 28. Error bars represent s.e.m. INF, days of infusion. Scale bars: 500 μm (A); 50 μm (B–G).
Fig. 4.
Fig. 4.
In vivo chronic exposure of the spinal cord to kainic acid – distal effects. (A) After 28 days of infusion (INF), NMJs treated with vehicle control and labelled for YFP (green) and α-bungarotoxin (red) were intact throughout the gastrocnemius muscle. (B) At 28 days of infusion with 10 mM kainic acid, NMJs labelled for YFP (green) and α-bungarotoxin (red) were shrunken. (C,D) NMJs were scored as either intact (C) or degenerating (D). (E) Quantitation of the percentage of degenerating synapses demonstrated a significant (*P<0.05) increase in the percentage of degenerating synapses in the mice that had been infused with 10 mM kainic acid for 14 and 28 days, in comparison with vehicle controls. Error bars represent s.e.m. Scale bars: 100 μm (A,B); 30 μm (C,D).
Fig. 5.
Fig. 5.
Forelimb and hindlimb motor function following 28 days infusion. (A) Motor performance was investigated using analysis of stride, sway and stance lengths for mice that had received 28 days of infusion of kainic acid or vehicle control into the spinal cord. (B) There was no significant difference between the stride, stance and sway lengths of the forelimb stride stance and sway lengths in the mice that had received kainic acid (KA) compared with those of animals receiving the vehicle control. (C) There was significantly (*P<0.05) reduced hindlimb stride, sway and stance length in kainic-acid-treated mice compared with those of animals treated with vehicle control. Error bars represent s.e.m.

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