Compact Myelin Detachment after metabolic Oligodendrocyte injury

Felix Schweiger1, Felix Fischbach1, Julia Nedelcu1, Friederike Pfeiffer2, Uta Chrzanowski1, Petra Fallier-Becker3, Markus Kipp1

1 Department of Anatomy II, Ludwig-Maximilians-University of Munich, 80336 Munich, Germany2 Group of Neuron Glia Interaction, Werner Reichardt Centre for Integrative Neuroscience, University of Tuebingen, 72072 Tuebingen, Germany

3 Institute of Pathology and Neuropathology, University of Tuebingen, 72076 Tuebingen, Germany

Our studies show a centrifugal progression of oligodendrocyte pathology during Cuprizone-induced metabolic injury. After initial oligodendrocyte cell body pathology, we propose that retraction of oligodendrocytes’ processes leads to retraction forces, resulting in CoMyD eventually paralleled by axonal injury. Future studies have to show whether CoMyD as well occurs in MS, and which factors regulate this process.

IntroductionMultiple sclerosis (MS) is characterized by demyelination and oligodendrocyte degeneration. The pathology of white matter injury induces neurodegeneration and in consequence accumulation of irreversible clinical disability. Mechanisms leading to oligodendrocyte and myelin degeneration are poorly understood, however centrifugal(early oligodendrocyte cell body pathology) and centripetal (early myelin sheath pathology) mechanisms have been described. Here we investigate structuralconsequences of Cuprizone-induced centrifugal oligodendrocyte degeneration.

Results

We would like to thank S. Wübbel, A. Baltruschat, S. Tost and B. Aschauer for their excellent technical assistance.

The author states no conflict of interest.

Butt, A. M., et al. (1998). "Axon-myelin sheath relations of oligodendrocyte unit phenotypes in the adult rat anterior medullary velum." Journal of Neurocytology 27(4): 205-217.

Romanelli, E., et al. (2016). "Myelinosome formation represents an early stage of oligodendrocyte damage in multiple sclerosis and its animal model." Nat Commun 7: 13275.

Kipp, M., et al. (2012). "Pathology of multiple sclerosis." CNS Neurol Disord Drug Targets 11(5): 506-517.

Kipp, M., et al. (2017). "Multiple sclerosis animal models: a clinical and histopathological perspective." Brain Pathol 27(2): 123-137.

Discussion

Literature

Centrifugal spread of oligodendrocyte damage in cuprizone lesionsDAPI ATF3 APC merge

control

4d Cup

1wk Cup

cells/m

m²

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ol

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up

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0 % ~78 %

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ATF3+-APC

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~17 % ~46 %

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0 % ~65 %

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APC+-cells

APC+-ATF3

+-cells

0 % ~62 %

Figure 1 (a) Immunofluorescence double labelling against the mature oligodendrocyte marker protein APC and the cellularstress response marker protein ATF3. Stars highlight cytoplasmic APC, whereas the arrows highlight APC+/ATF3+

oligodendrocytes. (b) Quantification of APC/ATF3-expressing cell populations after 4 days cuprizone intoxication. (c)Quantification of APC/ATF3-expressing cell populations after 1 week cuprizone intoxication. (d) Quantification of myelinproteins densities at week 1 and week 3. Arrows highlight apoptotic oligodendrocytes in d’. (e) Quantification ofdemyelination on the ultrastructural level at week 1. Results point to a sequence of damage that starts with degeneration ofoligodendrocyte followed by myelin loss (i.e., centrifugal oligodendrocyte pathology).

(a)

control 1 wk Cup

PLP

control

1 wk Cup

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(c)

(e)

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Figure 2 (a) Three-dimensional CoMyD reconstruction in 3D scanning electron microscopy image stacks(axon in orange, myelin in green). (b) Ultrastructural CoMyD appearance (axolemma in blue). (c)Quantification of different myelin pathologies at week 3. (c) Quantification of the spatial orientation of thedetached myelin sheaths. Arrowhead highlights an asymmetrical, whereas arrows highlight symmetricalmyelin detachment, respectively.

rela

tive f

req

uen

cie

s [

%]

CoM

yD

foca

l swel

ling

demye

linat

ion a

t NOR

spher

oid

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% o

f all C

oM

yD

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(b) (c)

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Figure 3 – (a) CoMyD numbers, analyzed in 3D scanning electron microscopy image stacks. (b) Quantification of axonaldiameters showing CoMyD pathology. (c) Quantification of CoMyD dimensions, analyzed in confocal images. (d)Representative confocal CoMyD image stacks. Sections were labelled using antibodies directed against the myelin marker PLP(red) and a neurofilament marker SMI-312 (green). (e) CoMyD numbers, analyzed in confocal image stacks.

CoMyD characteristics

nu

mb

er

per

mm

³

contr

ol

3 wks

Cup

0

5.0104

1.0105

1.5105

2.0105

***

IHC

qu

oti

en

t= h

/i

IHC

EM

0.4

0.6

0.8

1.0n.s.

3 wks Cup

nu

mb

ers

per

mm

³

contr

ol

3 wks

Cup

0

2.0106

4.0106

6.0106

8.0106

EMaxonal diameter (without myelin - in µm) - average of 6 measurements

freq

uen

cy (

%)

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3 wks Cup-CoMyD axons

control axons

(a) (b) (c)

(e)hi

m

(d)

Figure 4 – (a) Representative compound action potential (CAP) recordings, obtained from acute brain slicescontaining corpus callosum of control and 3 wks cuprizone intoxicated mice. N1 indicates myelinated axonsand N2 non-/de-myelinated axons. (b) Quantification of N1 and N2 CAP amplitudes . Note that the N2-Cuprizone response (green) does not reach the sum of N1N2-control response (purple).

Electrophysiology analysis of corpus callosum axons

(a)

(b)

myeli

n d

en

sity

(%

)

contr

ol

1 wk

Cup

3 wks

Cup

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3 wks

Cup

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Cup

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60

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CNPase MAG PLP

3 wks Cup1 wk CupHE

nu

mb

er

of

myelin

ate

d a

xo

ns/m

m²

contr

ol

1 wk

Cup

0

2.0105

4.0105

6.0105

8.0105

1.0106

Median with interquartil range

n.s.

Compact Myelin Detachment (CoMyD) at the beginning of activedemyelination (i.e., at week three)

(d‘)