D-Aspartate treatment stimulates differentiation of oligodendrocyte precursors, prevents demyelination and accelerates remyelination in the cuprizone mouse model of Multiple Sclerosis

Emerging evidence support a role for some D-Aminoacids as neurotrasmitters and neuromodulators, since they are found in mammalian tissues and also in the central nervous system (CNS) (Hashimoto and Oka, 1997). They play important roles in some physiological processes, including dendritic morphology, synaptic plasticity and cognition (Wolosker et al., 2008; Billard, 2012). Among D-Aminoacids, recent studies suggest that D-Aspartic acid (D-Asp), a newly discovered agonist for NMDA receptors, play a role in NMDA receptor-dependent processes such as synaptic plasticity and memory (Errico et al., 2015). The D-Asp was described in the multi-lamellar membrane that insulate axons (Fisher et al., 1986) and its effects on the hormone biosynthesis and release have been largely explored in the years (Gold and Voskuhl 2009; Nunez et al., 2000; Cerget et al., 2006). The exact mechanism of myelination process is still unknown, but emerging studies demonstrated the importance of intracellular changes in [Ca2+]i levels during myelination and remyelination processes (Soliven et al., 2001). Indeed, differentiation of oligodendrocyte precursors cells (OPC) and remyelination are associated with NMDARs-dependent [Ca2+]i changes (Martinez-Lozada et al., 2014). A recent work performed by our research group demonstrated that [Ca2+]i signaling mediated by the Na+/Ca2+ exchanger NCX3 plays an important role during oligodendrocytes differentiation and myelin formation (Boscia et al., 2012; Casamassa et al., 2016). In the present study, we investigated the effects of D-Asp during the OPC differentiation and remyelination by using both in vitro and in vivo techniques. In vitro, we evaluated the effects of D-Asp exposure both in human oligodendrocyte MO3.13 cell line and rat primary OPC, exposed to different concentrations of D-Aspartic acid (10-100-200 µM). Quantitative RT-PCR analyses showed that 10-200 μM D-Asp exposure for 3 days, upregulated, in a concentration-dependent manner, both the myelin markers CNPase and MBP and NCX3 transcripts in human oligodendrocytes M03.13 progenitors. The transcripts increase were significantly prevented by the NMDA receptor antagonist 10 µM MK-801 and the two NCX3 blockers, 30nM YM-244769 and 100nM BED. In accordance, microfluorimetric studies demonstrated that 100μM D-Asp administration induced an initial calcium peak of intracellular Ca2+ concentration [Ca2+]i followed by an oscillatory [Ca2+]i pattern both in oligodendrocyte MO3.13 progenitors and rat primary OPC. The NMDA antagonist 10µM MK-801 completly suppressed [Ca2+]i oscillations but only partially affected the first [Ca2+]i peak. Similar effects were observed in presence of the two selective blockers for NCX3, 30nM YM-244769 and 100nM BED. In addition, electrophysiological recordings performed in oligodendrocytes M03.13 progenitors showed that the current elicited by 100 µM D-Asp stimulation were dependent by AMPA activation, since the AMPA receptor inhibitor 10μM DNQX significantly prevented D-Asp induced inward currents. Our in vitro results suggest that D-Asp stimulates oligodendrocyte development through a mechanism involving calcium signaling through the glutamate receptors AMPA and NMDA and the Na+/Ca2+exchanger NCX3. Next, we investigated the effects of D-Asp administration in an in vivo model of demyelination/remyelination, the cuprizone mouse model. D-Asp was given during cuprizone feeding (demyelination), or after cuprizone withdrawal (remyelination). In both conditions, D-Asp treatment improved motor coordination performance in the beam balance and rotarod test. When given during demyelination D-Asp prevented MBP loss and reduced inflammation, as revealed by Western Blot analysis of MBP, Iba1 and GFAP proteins and quantitative coexpression analysis of MBP with the axonal marker NF200. Finally, electron microscopy performed on corpus callosum sections showed that D-Asp treatment accelerates remyelination in cuprizone mice, as demonstrated by the increased number in myelinated axons if compared to untreated cuprizone mice. Collectively, our results show that treatment with D-Aspartate, by influencing calcium signaling in oligodendrocytes, might produce beneficial effects during demyelination and remyelination processes.