ALS Patient-derived Oligodendrocytes Are Toxic to Motor Neurons Via Multiple Mechanisms

The case for a critical role for oligodendrocytes in ALS pathology just got stronger, based on evidence that stem cell-derived oligodendrocytes from ALS patients harm motor neurons through cell-to-cell contact and secretion of toxic substances. The results, reported in the October 18 PNAS, suggest that motor neuron damage can be reduced by shutting down SOD1 production even in oligodendrocytes derived from sporadic ALS patients, but not in those derived from C9ORF72-ALS patients. However, SOD1 shutdown needs to happen early; by the time the oligodendrocytes are mature, it’s too late.

“Once a mature oligo has adopted the toxic profile, once it is past that tipping point, you can’t reverse that,” commented senior author Brian Kaspar of Nationwide Children’s Hospital in Columbus, Ohio.

Brian Kaspar.

Brian Kaspar.

Previous studies in mutant SOD1 mice have implicated oligodendrocytes in motor neuron death (see Jan 2011 news, April 2013 news).  Kaspar, along with first author Laura Ferraiuolo, sought to investigate whether the same phenomenon occurred in human oligodendrocytes. In order to develop a reliable in vitro model for human ALS oligodendrocytes, second author Kathrin Myer developed an efficient differentiation protocol to generate oligodendrocytes from neural progenitor cells derived from either human iPS cells or by direct conversion of human fibroblasts (iNPCs). After a month of treatment in culture, 96% of surviving cells were positive for the oligodendrocyte marker myelin basic protein, and further gene expression analysis revealed enrichment for oligodendrocyte markers.

Next, the investigators co-cultured mouse motor neurons with either iPS-derived oligodendrocytes from ALS patients (three sporadic, one harboring a FIG4 mutation), or human induced neural progenitor cell (iNPC)-derived oligodendrocytes (four sporadic, three C9ORF72 mutation, one SOD1 mutation, one TDP43 mutation).  Within 72 hours, the motor neurons developed an abnormal phenotype, with axon shortening and “beading,” and death of between 40% and 60% of motor neurons within 11 days.

How might ALS patient-derived oligodendrocytes be exacerbating motor neuron death? In order to address this question, the investigators first tested the effect on motor neurons of conditioned media from either familial or sporadic ALS oligodendrocytes, and found that both types of media induced motor neuron death, an effect that could be reduced by dilution of the media.

Research over the past few years has revealed that, in additional to a role in myelination, oligodendrocytes secrete lactate, which is taken up by axons and used as an energy source (see July 2012 news). Furthermore, evidence from both human postmortem cortex and mSOD1 mouse spinal cord suggest that lactate transport is impaired in ALS and may contribute to neurodegeneration. In order to investigate the potential contribution of lactate to oligodendrocyte-induced toxicity, Kaspar and colleagues examined lactate secretion from the patient-derived oligodendrocytes. Intriguingly, they found no disease-related decline in lactate release early in oligodendrocyte differentiation, but levels were reduced compared to non-disease controls as the progenitor cells began to differentiate and express more oligodendrocyte-specific markers. The C9ORF72 patient-derived cells stood out, since they did not exhibit a decline in lactate production with maturation.

Can the toxic effects of ALS patient-derived oligodendrocytes be reversed? Lactate supplementation rescued motor neuron death caused by conditioned medium from both mouse mSOD1 and ALS patient-derived cells, again with the exception of C9ORF72 patients. But adding lactate was less effective when oligodendrocytes were in direct contact with motor neurons, indicating that survival “is likely mediated by both soluble and insoluble factors that require cell-to-cell contact or very close vicinity,” the authors wrote.

As lactate is a source of nutrition for motor neurons, the decline in production may indicate that oligodendrocytes “are struggling to keep up their metabolic activity in support of neurons,” Meyer said, though she added that this is likely only one of several pathways at work. Furthermore, she commented that, “The results also indicate that C9ORF72 cases seem to be distinct from other forms of ALS,” and may need different therapeutic strategies as a result.

This pattern of response to intervention early in differentiation, but not later, and differential response between C9ORF72 and other forms of ALS, was seen again when the team knocked down human SOD1 expression in oligodendrocytes in each of the ALS lines. Remarkably, both sporadic and familial ALS-derived oligodendrocytes, with the exception of C9ORF72 lines, were much less toxic to motor neurons following treatment with shRNA against SOD1.  Misfolded SOD1 may be implicated as a common contributor to toxicity in the cell lines that responded. This hypothesis was supported by detection of misfolded SOD1 in oligodendrocytes derived from patients with sporadic ALS, as well as with ALS due to mutations in SOD1, TDP43, and FIG4, but not C9ORF72.

Dwight Bergles.

Dwight Bergles.

The results of this study provide further support for a role for oligodendrocytes in ALS pathogenesis, commented Dwight Bergles of Johns Hopkins University in Baltimore, Maryland, who was not involved in the study. “Our postmortem analysis of brain tissue from ALS patients revealed that demyelination was prevalent in cortical gray matter in both sporadic and familial forms of ALS. The studies by Ferraiuolo et al., corroborate key aspects of these findings using human cells. They also extend our findings by showing that in some forms of familial and sporadic ALS, the toxic properties of oligodendrocytes involve SOD1, even when this gene is not mutated. Together, this body of work reinforces the conclusion that oligodendrocyte dysfunction accelerates motor neuron death in some forms of ALS.”

He cautioned, however, that it may be premature to conclude that C9ORF72 operates through entirely distinct mechanisms. “One caveat is that these experiments were performed in vitro, and it is possible that additional stresses may be placed on oligodendrocytes in the intact CNS, particularly as disease progresses and inflammation become prevalent. Although the death of motor neurons can be studied in vitro, it is not clear that this form of death is comparable to the progressive degeneration of mature motor neurons in vivo. Additional support for this conclusion may come from analysis of oligodendrocyte behavior in new mouse models of C9 ALS.”

Noting that some motor neurons were spared in the assays, Bergles suggested that analysis of these cells could provide clues to both the selective protection of certain motor neurons and the mechanisms by which mutant oligodendrocytes induce motor neuron death.

Ben Barres.

Ben Barres.

Ben Barres of Stanford University in California, who was not involved in the work, was more cautious about the conclusions drawn from the study, because the oligodendrocyte cultures used still had a small percentage of other cell types in them, likely including astrocytes, which are known to be toxic (see Aug 2011 newsFeb 2014 news). “I am not saying that the oligodendrocyte lineage cells are not making a toxin, but to prove this one would either have to use pure oligodendrocyte cultures, or identify the toxin and show that it is actually expressed by the oligodendrocytes. I am entirely open to the possibility that other cell types besides astrocytes may also secrete toxic substances under the right conditions, but this needs to be shown convincingly with purified cell types, given that it is already so well established that mutant SOD1 astrocytes can be so highly toxic.”

Primary Reference:

Ferraiuolo L, Meyer K, Sherwood TW, Vick J, Likhite S, Frakes A, Miranda CJ, Braun L, Heath PR, Pineda R, Beattie CE, Shaw PJ, Askwith CC, McTigue D, Kaspar BK. Oligodendrocytes contribute to motor neuron death in ALS via SOD1-dependent mechanism. Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):E6496-E6505.[Pubmed].

Home page image: Wikimedia Commons.

disease-als oligodendrocytes SOD1 topic-preclinical
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