Involvement of Peripheral Nerves in the Transgenic PLP-α-Syn Model of Multiple System Atrophy: Extending the Phenotype

Abstract

Multiple system atrophy (MSA) is a fatal, rapidly progressive neurodegenerative disease with (oligodendro-)glial cytoplasmic α-synuclein (α-syn) inclusions (GCIs). Peripheral neuropathies have been reported in up to 40% of MSA patients, the cause remaining unclear. In a transgenic MSA mouse model featuring GCI-like inclusion pathology based on PLP-promoter driven overexpression of human α-syn in oligodendroglia motor and non-motor deficits are associated with MSA-like neurodegeneration. Since α-syn is also expressed in Schwann cells we aimed to investigate whether peripheral nerves are anatomically and functionally affected in the PLP-α-syn MSA mouse model.

Results: To this end, heat/cold as well as mechanical sensitivity tests were performed. Furthermore, in vivo and ex vivo nerve conduction and the G-ratios of the sciatic nerve were analyzed, and thermosensitive ion channel mRNA expression in dorsal root ganglia (DRG) was assessed. The presence of human α-syn in Schwann cells was associated with subtle behavioral impairments. The G-ratio of the sciatic nerve, the conduction velocity of myelinated and unmyelinated primary afferents and the expression of thermosensitive ion channels in the sensory neurons, however, were similar to wildtype mice.

Conclusion: Our results suggest that the PNS appears to be affected by Schwann cell α-syn deposits in the PLP-α-syn MSA mouse model. However, there was no consistent evidence for functional PNS perturbations resulting from such α-syn aggregates suggesting a more central cause of the observed behavioral abnormalities. Nonetheless, our results do not exclude a causal role of α-syn in the pathogenesis of MSA associated peripheral neuropathy.

Fig 1. Experimental design.

Fig 1 illustrates experimental design and gives an overview of the behavioral tests performed at each time-point (5–15 months) and which days (D1—D10).

Fig 2. Behavioral characterization of tg PLP-α-syn mice and age-, gender- and background-matched wt controls at different time-points.

Investigation of the motor abilities (A) measured as the time spent on the rotarod and mechanical sensitivity (B) indicated by the paw-withdrawal-threshold (PWT) did not reveal any differences between the groups at any time-point. The Hargreaves test for heat sensitivity did not show any significant difference in the paw-withdrawal-latency (PWL) between tg PLP-α-syn and wt animals, however, all values of the tg group were lower than the wt group indicating a trend towards increased heat-sensitivity (C). Analysis of the tailflick test revealed increased heat sensitivity in tg PLP-α-syn animals at 12 and 15 months of age (D) supporting the trend seen in the Hargreaves test. The cold plate-test revealed increased sensitivity to cold stimuli in tg PLP-α-syn animals at 12 and 15 months as seen by the decreased delay of the first jump (E) at 12 months and the increased number of jumps (F) at 12 and 15 months. n = 8, data represent mean values ± S.E.M.

Fig 3. In vivo and ex vivo measurement of nerve conduction velocity.

In vivo sciatic nerve conduction velocity was measured in 12 month-old tg PLP-α-syn and wt animals (A). No difference was detected in the conduction properties between the groups. n = 8. Sciatic nerve conduction velocity was measured ex vivo in 15 month-old tg PLP-α-syn and wt animals. No difference was detected in the conduction velocity (B) or stimulus strength required (C) between the groups. n = 3, data blotted represent mean values ± S.E.M.

Fig 4. Immunofluorescence stainings of tg sciatic nerve sections stained with the hα-syn marker 15G7 and the Schwann cell marker CNPase.

The arrows highlight the co-localization pattern of h—syn (first column, images A, D, G, J) and the Schwann-Cell marker CNPase (second column, images B, E, H, K) in the tissue of tg PLP-α-syn animals, the third column shows the co-labeling of h—syn and CNPase and includes a staining of the nuclei with DAPI (images C, F, I, L). Images D, E, F show magnified areas of A, B, C and J, K, L show magnified sections of G, H, I. scale bar = 10 μm.

Fig 5. Immunofluorescence stainings of tg sciatic nerve sections stained with the hα-syn marker 15G7 and the Schwann cell marker S-100.

The arrows highlight the co-localization pattern of h—syn (first column, images A, D, G) and the Schwann-Cell marker S-100 (second column, images B, E, H) in the tissue of tg PLP-α-syn animals (last 2 rows, images D-I) and wt animals as negative control for the h—syn antibody (first row, images A-C), the third column shows the co-labeling of h—syn and S-100 and includes a staining of the nuclei with DAPI (images C, F, I). Images G, H, I show magnified areas of D, E, F. scale bar = 10 μm.

Fig 6. Immunofluorescence labeling of wt and tg DRG sections with the neuronal marker β-III-tubulin and the hα-syn marker 15G7.

DRG tissue of wt and tg PLP-α-syn animals stained with the neuronal marker β-III-tubulin (red), the marker for hα-syn 15G7 (green) and DAPI (blue) to label the nuclei. This image confirms the presence of hα-syn in the DRG of the PLP-α-syn mouse model. It can be seen, that the green hα-syn is located around the neurons in red and the markers do not co-localize; scale bar = 10 μm.

Fig 7. G-ratio of 15 month-old wt and tg PLP-α-syn sciatic nerve sections.

The investigation of myelin thickness by calculating the G-ratio (inner axon diameter/outer axon diameter) did not detect differences between tg PLP-α-syn and wt animals in axons thinner than 5 μm, axons between 5 μm and 8 μm and axons between 8 μm and 12 μm (p = 0.1095). Ultra-thin sections of wt and tg PLP-α-syn sciatic nerve tissue have been labeled with toluidine blue, the measurements were performed in microphotographs with 100x magnification. n = 3 animals, 300 fibers of equal distribution analyzed per animal, data represent mean values ± S.E.M.

Fig 8. No difference in mRNA expression levels of Trpa1 and Trpm8 between wt and tg MSA mice.

Trpa1 and Trpm8 mRNA levels were similar for wt and tg PLP-α-syn DRG explants. n = 5, Boxes represent median as well as lower and upper quartile; whiskers represent the 10th and 90th percentiles, the mean value is indicated with the ‘+’ symbol.