While there are many highly effective treatments reducing inflammatory activity and relapse rate in MS, therapies that promote myelin repair are still unavailable. Tapping into the regenerative potential of cell reservoirs such as OPCs or adult neural stem cells (NSCs) therefore represents one of the biggest currently unmet clinical needs [
29]. In order to achieve remyelination, two major approaches are conceivable: The achievement of either an exogenous neutralization of inhibitory elements or the implementation of a direct stimulation of regenerative mechanisms [
30]. To this end, new drugs will have to be developed or already approved drugs with regenerative properties will have to be repurposed. The past 10 years have seen tremendous progress in both fields as evidenced by the clinical trials evaluating the anti-LINGO1 antibody opicinumab [
31] or the antihistamine/anticholinergic drug clemastine [
32] for efficacy in MS and optic neuritis (ON), respectively. However, with the opicinumab trial not meeting its primary endpoint [
31] and clemastine only achieving limited effects, the search for regeneration-conferring substances is ongoing [
29].
In the present study, we demonstrate that teriflunomide induces transcriptional regulators required for proper oligodendroglial cell differentiation such as Mash1/Ascl1, Sox 10, Myrf [
23], Nkx2.2 [
24,
25], and TAp73 and increases myelin expression when OPCs are exposed to timed pulses of the substance early during spontaneous differentiation. This observation was corroborated by a change in the subcellular localization of p57kip2 in stimulated cells, which we had previously identified as a regulator of oligodendroglial differentiation competence [
21,
33]. Of note, such pro-oligodendroglial teriflunomide concentrations did not significantly reduce cell survival as opposed to higher concentrations. We confirmed our results in myelinating neuron/glia co-cultures demonstrating that early teriflunomide pulses also increase internode formation by oligodendroglial cells. Of note, in this more complex culture paradigm, an extended (constant) teriflunomide application did not decrease myelination indicating that prolonged application as occurring in the context of the RRMS therapy is most probably not counterproductive to repair. Mechanistically, we could demonstrate that long-term application of teriflunomide for 3 days led to a downregulation of CRM1 and Nkx2.2, the latter of which was shown to cooperate with Mash1/Ascl1 [
28], and direct binding to corresponding regulatory regions was demonstrated to result in activation of myelin related genes such as ceramide galactosyltransferase [
34,
35], PLP [
24], and MBP [
36]. In addition, experiments in
Nkx2.2-null mutant mice revealed that the differentiation of MBP- and PLP/DM20-positive oligodendrocytes is dramatically retarded [
25], corresponding to our gene expression (Figs.
2 and
6) and protein expression data (Fig.
4). On the other hand, downregulation of CRM1, which is relevant for the shuttling of p57kip2 protein from the nucleus into the cytoplasm [
21], hence also affects the activity of the pro-oligodendroglial transcription factor Mash1/Ascl1. Of note, early teriflunomide pulses significantly boosted Myrf transcript levels, a transcription factor essential for myelination during development but also shown to contribute significantly to myelin repair [
37]. Moreover, the transcription factor TAp73 was found to be upregulated by teriflunomide stimulation specifically in immature OPCs while mature cells appeared to be unaffected. Whether this is due to a lowered sensitivity of matured cells towards teriflunomide or whether additional signaling cascades are initiated later on which neutralize or counteract pro-oligodendroglial subcellular processes remains to be shown by future studies. TAp73 belongs to the p53 superfamily of transcription factors, which, despite their strong homology, have acquired a high degree of functional specificity. TAp73 seems to be highly relevant in neurogenesis as respective knock-out mice feature, among other abnormalities, hippocampal dysgenesis and hydrocephalus [
38]. Moreover, increased tumorigenesis as demonstrated in p53 knock-out animals is not observed [
38]. Furthermore, teriflunomide was shown to have an inhibitory impact on store-operated Ca
2+ influx (SOCE)-mediated calcium signaling [
10] which is relevant for OPC proliferation [
39,
40] explaining the observed reduction of Ki67-positive OPCs (Fig.
1).
Of note, teriflunomide concentrations used in our experiments were matched to the concentrations reaching the brain during orally administered therapy (2.5–4.1 μM) at least during inflammatory relapses where a collapse of the blood-brain barrier (BBB) can be observed [
16,
17,
41]. While this study demonstrated that beyond its well-described effect on inflammation via targeting the proliferation of activated lymphocytes, teriflunomide can also modulate OPC differentiation and myelination; it also showed that there is a specific window of opportunity for such modulation. In how far these effects can be harnessed to contribute to neurorepair in MS, potentially in the context of transiently elevated teriflunomide levels in response to BBB impairment, remains to be elucidated. Finally, pulsed teriflunomide applications might be feasible in patients under other neuroimmunological treatments and may thus provide an add-on effect on tissue restoration.
Furthermore, short-term teriflunomide treatment could also be beneficial in other CNS diseases with white matter damage such as amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), or Alzheimer’s disease (AD; [
30]) when applied during specific disease stages. Future studies in inflammatory and non-inflammatory CNS disease models must therefore be conducted using different application schemes in order to clarify teriflunomide’s potential as a regenerative compound for biomedical translation. In this regard, it will also be highly relevant to confirm that an extended teriflunomide application cannot harm basic myelination levels in vivo.