Cell Fusion along the Anterior-Posterior Neuroaxis in Mice with Experimental Autoimmune Encephalomyelitis

Abstract

Background: It is well documented that bone marrow-derived cells can fuse with a diverse range of cells, including brain cells, under normal or pathological conditions. Inflammation leads to robust fusion of bone marrow-derived cells with Purkinje cells and the formation of binucleate heterokaryons in the cerebellum. Heterokaryons form through the fusion of two developmentally differential cells and as a result contain two distinct nuclei without subsequent nuclear or chromosome loss.

Aim: In the brain, fusion of bone marrow-derived cells appears to be restricted to the complex and large Purkinje cells, raising the question whether the size of the recipient cell is important for cell fusion in the central nervous system. Purkinje cells are among the largest neurons in the central nervous system and accordingly can harbor two nuclei.

Results: Using a well-characterized model for heterokaryon formation in the cerebellum (experimental autoimmune encephalomyelitis - a mouse model of multiple sclerosis), we report for the first time that green fluorescent protein-labeled bone marrow-derived cells can fuse and form heterokaryons with spinal cord motor neurons. These spinal cord heterokaryons are predominantly located in or adjacent to an active or previously active inflammation site, demonstrating that inflammation and infiltration of immune cells are key for cell fusion in the central nervous system. While some motor neurons were found to contain two nuclei, co-expressing green fluorescent protein and the neuronal marker, neuron-specific nuclear protein, a number of small interneurons also co-expressed green fluorescent protein and the neuronal marker, neuron-specific nuclear protein. These small heterokaryons were scattered in the gray matter of the spinal cord.

Conclusion: This novel finding expands the repertoire of neurons that can form heterokaryons with bone marrow-derived cells in the central nervous system, albeit in low numbers, possibly leading to a novel therapy for spinal cord motor neurons or other neurons that are compromised in the central nervous system.

Fig 1. Immune cell infiltration and heterokaryon formation in EAE mouse cerebella.

(A) Comparison between coronal sections of cerebella from Control (left) and EAE immunized mice (right). There is a higher number of GFP-labeled infiltrating bone marrow-derived cells and Purkinje heterokaryons (arrows) in EAE affected mice as compared to control animals. Scale bar 300 μm. (B) Quantification of Purkinje heterokaryons in Control (n = 3; 1.0 ± 0.6) and EAE (n = 7; 77.7 ± 16.7) shows a significant difference (p = 0.0167, Mann-Whitney-Wilcoxon test). (C) In EAE, more heterokaryons were located in the vermis (54.4 ± 12.0) than in the lateral hemispheres (n = 7; 23.3 ± 5.0) (n = 7, p = 0.0313).

Fig 2. Immune cell infiltration in the spinal cord of Control and EAE affected mice.

Coronal spinal cord sections showed little infiltration of GFP-labeled bone marrow-derived cells (green) in control (left) animals, while infiltration in EAE immunized animals (right) was prominent. Scale bar 300 μm.

Fig 3. Formation of a heterokaryon in the spinal cord.

(A) A motor neuron that is present in the ventral horn of the spinal cord expresses GFP (arrow) as a result of fusion between a GFP expressing bone marrow-derived cell and a motor neuron. The arrow shows a single GFP-labeled motor neuron in the ventral horn of the spinal cord. Scale bar 300 μm. (B) Higher magnification of a spinal cord GFP-labeled motor neuron shown in (A). Scale bar 150 μm. (C-E) Z-stack images of the GFP-labeled motor neuron shown in (A) and (B). (D-E) Immunohistochemistry demonstrating that the GFP-labeled motor neuron co-expresses GFP and NeuN. (F) Triple staining of the same motor neuron, NeuN (red), GFP (green) and Hoechst (blue). Two nuclei (Hoechst, blue) are present in the same cell, marked with dotted circles thus it is a heterokaryon. Scale bar 25 μm.

Fig 4. GFP-labeled spinal cord motor neuron co-expressing NeuN.

(A) GFP-labeled (green) ventral horn motor neuron (arrow) extending a single axon from the grey matter (GM) to the white matter (WM) (see arrowheads). (B-C) This GFP-labeled motor neuron co-expresses NeuN. (D-F) Higher magnification of the motor neuron in B-C. Scale bar (A-C) 150 μm, (D-F) 25 μm.

Fig 5. Quantification and distribution of heterokaryons in EAE affected and Control spinal cord.

(A) While there was a wide spread in the number of heterokaryons between individual EAE affected mice (n = 5; 20.0 ± 6.7), depending on the severity of inflammation, there were significantly more heterokaryons (p = 0.0358, Mann-Whitney-Wilcoxon test) in EAE affected mice than Control (n = 3; 0.3 ± 0.3) mice. (B) Schematic representation of the distribution of heterokaryons in 20 sections of EAE spinal cord. Each symbol represents one experimental animal, and the symbol size represents heterokaryon size (small symbol: <20 μm, large: >20 μm).

Fig 6. GFP-labeled motor neurons and interneurons in the spinal cord.

(A) In a coronal section of the spinal cord with prominent infiltration of GFP-labeled bone marrow-derived cells (green), a number of GFP-labeled interneurons were detected. (B) Magnification of the area indicated with a box in (A). Eight GFP-labeled interneurons (green, indicated by arrows) located in the grey matter (GM), and one motor neuron (arrowhead) extending an axon/dendrites across the white matter (WM). (C) Most of these cells co-label with NeuN (indicated by arrowheads). (D) Schematic illustration of GFP-labeled interneuron and their dendrites. Scale bar (A-D) 150 μm.