PLoS One. 2017 Jun 5;12(6):e0178622. doi: 10.1371/journal.pone.0178622. 

Acid sphingomyelinase deficiency enhances myelin repair after acute and chronic demyelination.

Chami M1, Halmer R1, Schnoeder L1, Anne Becker K2, Meier C3, Fassbender K1, Gulbins E2,4, Walter S1,5,6.

1 Department of Neurology, Saarland University Hospital, Homburg, Germany.
2 Department of Molecular Biology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
3 Institute of Anatomy and Cell Biology, Saarland University, Homburg, Germany.
4 Department of Surgery, University of Cincinnati, Cincinnati, Ohio, United States of America.
5 Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia.
6 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia.




MS is a chronic inflammatory disease of the CNS characterized by inflammatory infiltrates, demyelination and axonal loss. It is the most frequent cause of neuro-logical disability in young adults (Lassmann et al., 2001). The invasion of lym-phocytes leads to a subsequent myelin loss, astrocyte and microglia activation and oligodendrocyte apoptosis. Recovery and remyelination are challenged by several factors such as the blockage of the proliferation and differentiation of OPCs required to repair damaged myelin (Gudi et al., 2014).

In this study, we demonstrate that Asm deficiency enhances myelin repair after acute or chronic experimental demyelination by lowering astrocytosis and with a significant increase of oligodendrocyte proliferation. Thus, Asm plays a crucial role in the pathophysiology of MS both acting on T cell transmigration and enhancing remyelination by indirect effect on astrocytes and increased oligodendrocyte proliferation and differentiation. Thus, the Asm/ceramide system can enhance remyelination and recovery after cuprizone induced demyelination.

The results of this study demonstrate that genetic deficiency or pharmacological inhibition of the Asm/ceramide system can enhance myelin repair after cuprizone induced demyelination. Asm and its enzymatic product ceramide have both been shown to be higher expressed in active human MS lesions (Kim et al., 2009; van Doorn et al., 2010). Especially, the role of ceramide can be detrimental. By deficiency of the Asm/ceramide system, we could observe a significantly improved recovery of myelin as indicated by MBP and LFB intensity scores and an increase in oligodendrocyte proliferation as indicated by in-creased Olig-2 positive cells in the recovery phase after cuprizone treatment. This led to a reduction of neuronal damage. Interestingly, MBP/LFB levels and Olig-2 positive cell numbers were close to normal values of completely untreat-ed mice. This is in accordance with the observation that ceramide has been shown to induce oligodendrocyte cell death together with the pro-inflammatory cytokine tumor necrosis factor alpha in human MS brains. In addition, overexpression of Asm and subsequent increase of ceramide resulted in reduced neuronal survival even without any further cell stress (Kim Hye et al., 2011; Kim SunJa et al., 2012; Gulbins et al., 2013).

Fibroblast growth factors are important drivers of myelination (Mohan et al., 2014; Furusho et al., 2012; Messersmith et al., 2000). Further supporting the beneficial effect of Asm/ceramide deficiency, FGF1 and FGF2 showed a significant increase in the recovery phase after acute demyelination. Activation of CNS cells, especially astrocytes, is discussed as important mechanism in MS associated de- and remyelination. Astrocytes can show a dual role with either beneficial functions by facilitating e.g. microglial-mediated myelin clearance (Skripuletz et al., 2012) or detrimental functions by enhancing inflammatory myelin toxic reactions by e.g. inflammatory cytokine production (Gudi et al., 2014). Asm-induced ceramide is the principle component of ceramide-enriched mem-brane platforms, which are a pre-requisite for full cell activation facilitating membrane receptor assembling and subsequent intracellular signalling with possible thunder storm of pro-inflammatory mediators (Gulbins and Li, 2006).

In the current project, as consequence of the Asm genetic deficiency and sub-sequent reduced ceramide level, a significant reduction of activated astrocytes and the pro-inflammatory cytokine IL-1β could be observed and might act as potential mechanism for the optimized myelin repair. However, astrocyte numbers in Asm deficient mice are significantly, but only 30%, reduced compared to WT littermates. This is important as a complete ablation of astrocytes leads to a deprivation of their beneficial functions (Skipuletz et al., 2013). The observed differences upon Asm deficiency were seen in the recovery phase after acute demyelination with 5 weeks of cuprizone treatment, hinting towards an involvement of glial cells mainly after acute myelin injury. Recovery after chronic de-myelination (12 weeks of cuprizone) resulted in a still increased myelin content and oligodendrocyte proliferation, but did not show a clear glial cell involvement. There was also no difference in the extent of acute or chronic demyelination directly after 5 or 12 weeks of cuprizone treatment, indicating an equal mode of action of cuprizone in both, genetically deficient mice or wt littermates and fur-ther demonstrating a relevant effect of the Asm/ceramide system in promotion of remyelination rather than augmentation of demyelination.

Microglia are reported to play a supportive role in remyelination (Gudi et al., 2014). In the cuprizone animal model, they act as marker for drug-induced pathology and are increased and activated after cuprizone treatment (Goldberg et al., 2015). Interestingly, we did not see any difference in these increased micro-glia cell numbers or in their activation in Asm-deficient versus WT littermates in the recovery after acute demyelination, excluding any important role of microglia in myelin repair.


Pharmacological inhibition of Asm and myelin repair

As potential therapeutic approach to support remyelination, we could show that the positive effect of Asm deficiency in myelin recovery can be mimicked by treatment with amitriptyline, a well-known pharmacological Asm inhibitor (Kornhuber et al., 2010). Amitriptyline treatment was only initiated after cuprizone-induced myelin damage and was able to restore MBP and LFB intensity to nor-mal values after 2 weeks with an even higher than normal level of oligodendro-cyte proliferation. Again astrocytes were inhibited as potential mechanism for neuronal repair. These results strongly hint towards a detrimental role of the Asm/ceramide system not during myelin damage but during potential repair mechanisms.

We show that acid sphingomyelinase/ceramide deficiency or amitriptyline-induced pharmacological inhibition enhance remyelination after acute and chronic myelin damage. As potential mechanism a reduction in astrocyte proliferation and activation with reduced pro-inflammatory cytokine release, resulting in enhanced oligodendrocyte proliferation and neuronal preservation could be observed.

Using the toxic demyelination model, we wanted to investigate the role of Asm both in demyelination and as well as in the remyelination phase. During demyelination no results suggested any significant role for Asm, results showed no significance difference between Asm genetically deficient mice and wildtype mice. In contrast to demyelination, in remyelination we reported a significance difference in myelin repair after two weeks of the cuprizone withdrawal with increased oligodendrocyte proliferation in the Asm defi-cient mice compared to wildtype.

Hence, our data point towards an important role of Asm in the remyelination phase both after acute and chronic demyelination. To further investigate and to confirm the role of Asm, we used the functional inhibitor amitriptyline in the 2 weeks recovery phase after cuprizone withdrawal to study whether the pharma-cological inhibition of amitriptyline could yield to similar results as obtained from the Asm deficient mice in the cuprizone model. Amitriptyline injected mice in the recovery phase showed a significant myelin repair compared to mice injected with PBS at the same period and phase of recovery. With results corresponding to the genetic deficiency of Asm, increased oligodendrocte proliferation was documented with a lower degree of astrocyte activation.

Since the BBB remains intact in the cuprizone model we excluded the effect of Asm on T cells in the toxic demyelination model, and focused our interest on the glial cells. As documented Asm inhibition did significantly decrease astrogliosis in the 2 weeks recovery phase with a higher oligodendrocyte proliferation and differentiation, but did not show any effect on microgliosis.

Might be that it is affecting the remyelination pathway which microglia does not have a crucial role in. Certainly, the 30% decrease in astrogliois compared to that of wildtype has a beneficial effect on remyelination as was suggested before that astrocytes troubles remyelination in chronic MS by inhibiting OPCs proliferation and differentiation. Therefore, the inhibition of Asm enhanced re-myelination by an in-direct effect on astrocytes and oligodendrocyte proliferation.

We report the role of Asm in remyelination showing enhanced myelin recovery after acute and chronic demyelination. With respect to these findings Asm-ceramide system might be a therapeutic option for MS patients.



Fig 1. Immunohistochemical staining and quantification of brain sections using LFB and MBP. Cuprizone treatment for 5- or 12-weeks causes a massive decrease in myelination as determined by LFB and MBP staining in the CCm (a). Asm-deficient mice showed a significantly increased remyelination compared to wild-type littermates after 2 weeks of recovery following cuprizone treatment as determined by the quantification of myelination score (b) and MBP fluorescence intensity in the CCm (c). Representation of the corpus callosum (d).*p<0.05 Asm-deficient vs wild-type, scale bar: 100 μm.



Fig 2.Olig-2 staining for different treatment groups and mRNA analysis of FGF-1 and FGF-2 at week 7. Olig-2+ cell numbers were significantly increased in Asm-deficient mice compared to wild-type after acute or chronic cuprizone treatment followed by 2 weeks recovery period (a and b). Arrow indicating a strong expression of Olig-2 stain referring to an OPC and nearby a weakly expressed stain by a mature oligodendrocyte. FGF-1 and FGF-2 mRNA levels were also significantly increased after 2 weeks of recovery (week 7) in Asm-deficient mice compared to wild-type littermates (c and d).*p<0.05 Asm-deficient vs wild-types. Scale bar: 100μm.



Fig 3. Effect of Asm-deficiency on astrogliosis. GFAP stainings in the CCm after 5 or 12 weeks cuprizone treatment followed by 2 weeks remyelination. A significant decrease of astroglial distribution in Asm-deficient compared to wild-type littermates after 5 weeks of cuprizone treatment followed by 2 weeks of remyelination was observed (b and c). Asm-deficiency significantly decreased Il-1ß mRNA expression levels compared to wild- type littermates after 5 weeks cuprizone treatment followed by 2 weeks recovery (d). *p<0.05 Asm-deficient vs wild-type littermates. Scale bar: 100 μm.





Fig 4.Representative brain sections with CCm stained for LFB and MBP of amitriptyline-treated mice and respective controls. The extent of de- and remyelination was assessed by scoring LFB (a) stained brain section and quantifying MBP (b) intensity of the CCm. The inhibition of Asm by amitriptyline significantly restored myelin compared to control-treated animals after 5 weeks cuprizone treatment followed by 2 weeks of recovery (c and d). ***p<0.001 and *p<0.05 amitriptyline-treated vs control-treated littermates. Scale bar: 100 μm.



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