Control of local protein synthesis and initial events in myelination by action potentials

Abstract

Formation of myelin, the electrical insulation on axons produced by oligodendrocytes, is controlled by complex cell-cell signaling that regulates oligodendrocyte development and myelin formation on appropriate axons. If electrical activity could stimulate myelin induction, then neurodevelopment and the speed of information transmission through circuits could be modified by neural activity. We find that release of glutamate from synaptic vesicles along axons of mouse dorsal root ganglion neurons in culture promotes myelin induction by stimulating formation of cholesterol-rich signaling domains between oligodendrocytes and axons, and increasing local synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn kinase-dependent signaling. This axon-oligodendrocyte signaling would promote myelination of electrically active axons to regulate neural development and function according to environmental experience.

Fig. 1

Release of synaptic vesicles from axons promotes myelination. (A) Synaptic vesicle release from DRG neurons was blocked by adding BnTX or TnTX to neuron cultures, and OPCs were added after washing out the toxin. Five days later, axons were stimulated for 5 hours (10 Hz, 9 s at 5-min intervals), and they were examined 21 days later. (B and C) Myelin formation was greatly reduced in cultures in which vesicular release was blocked (MBP, green; neurofilament, purple). Scale bar, 10 μm (P < 0.005, n = 7). (D and E) OPCs had differentiated into oligodendrocytes regardless of whether vesicular release was blocked during electrical stimulation (black bar, −BnTX; gray bar, +BnTX), as indicated by protein expression (D and E) for myelin proteins [proteolipid protein 1 (PLP1), MBP, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP)] and the transcription factor Olig2.

Fig. 2

Electrical activity in axons is signaled to OPCs by the neurotransmitters glu and ATP. The two neurotransmitters are released through different mechanisms and produce different spatiotemporal Ca²⁺ responses in OPCs. (A) Ca²⁺ responses were seen in the cell body of OPCs (white arrows), using the Ca²⁺ indicator Oregon Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 1, AM ester form (BAPTA-1 AM), in response to electrical stimulation of cocultures when synaptic vesicle release was blocked with BnTX, but responses in OPC cell processes (green box and arrows) were only seen when vesicular release was not blocked. Scale bar, 10 μm. (B) Plot of Ca²⁺ responses in the soma (red) and cell processes (black) of OPCs shown in (A). Note the absence of responses in OPC cell process on neurons when vesicular release was blocked with BnTx. Red bar, 10 Hz field stimulation. (C) No Ca²⁺ response was produced when action potentials were blocked with tetrodotoxin (TTX), or when stimulation was delivered to OPCs in monoculture (*P < 0.005, n = five dishes in each category). (D) The peak Ca²⁺ concentration and (E) rate of Ca²⁺ rise after stimulation were not statistically different in the cell body of OPCs (red) on neurons treated with BnTX. No Ca²⁺ responses were evident in the cell processes (black) after stimulating neurons treated with BnTX (n = 13 dishes for each condition). (F) Somatic Ca²⁺ responses were inhibited by the P2 receptor blocker suramin (50 μM) and were completely blocked by a combination of suramin and glu receptor antagonists AP5 (50 μM) and CNQX (20 μM). (G) The genetic Ca²⁺ reporter GCaMP2 transfected into OPCs (green) enabled Ca²⁺ responses to be measured specifically in OPCs. Immunological staining of chondroitin sulfate proteoglycan 4 (NG2, red) and platelet-derived growth factor receptor (PDGFR) (purple). (H) Ca²⁺ responses were not seen when GCaMP2 was used in OPC cell processes and vesicular release was blocked by BnTX. (I) Summary data showing Ca²⁺ responses measured by GCaMP2. Glu antagonists were 20 μM CNQX + 50 μM AP5 + 500 μM MCPG [**P < 0.001 (black bar); n = 45 cells.] Ca²⁺ responses after 5 hours of stimulation were similar to acute responses (fig. S8).

Fig. 3

Formation of axon-oligodendrocyte signaling domains by electrical stimulation. Trafficking of TfR into cholesterol-rich membrane domains of OPCs was increased by the vesicular release of glu from neurons induced by electrical stimulation. (A) Punctate expression of TfR on the membrane (yellow) was greatly increased by 5 hours of electrical stimulation (10 Hz, 9 s, at 5-min intervals) of neurons (purple, neurofilament), but BnTX treatment or glu receptor antagonists (20 μM CNQX + 50 μM AP5), strongly inhibited trafficking of TfR into the membrane induced by electrical stimulation. Scale bar, 10 μm. (B) Stimulation of neurons treated with BnTX or in the presence of glu receptor antagonists inhibited trafficking of TfR receptor into cholesterol-rich membrane domains after stimulation (*P < 0.005; **P < 0.001; n = 21 cells from 21 dishes in each condition). (C) Phosphorylated Fyn kinase (red) colocalized with TfR receptor (green), consistent with axo-glial signaling localized at cholesterol-rich microdomains. (D) Electrical stimulation increased phosphorylation of Fyn kinase (p-Fyn), and this was blocked by BnTX treatment.

Fig. 4

Action potentials induce local translation of MBP by vesicular release of glu. (A) Experiments were carried out on rat OPCs before expressing MBP protein, although MBP mRNA was present. (B) Local translation of MBP was studied by transfecting OPCs with kikume protein. Actinomycin D was used to block transcription and the appearance of newly synthesized green-fluorescent MBP was monitored. (C) Electrical stimulation induced local translation in OPCs transfected with kikume-MBP-3′UTR (white arrows), which was inhibited by pre-treating axons with BnTX or stimulation in the presence of glu receptor antagonists (20 μM CNQX + 50 μM AP5 + 500 μM MCPG) (see also fig. S10). Scale bar, 10 μm. Pixel intensity is shown on an 8-bit pseudocolor scale. (D) Statistical analysis shows that the local translation was strongly increased by electrical stimulation and that blocking vesicular release with BnTX significantly decreased stimulus-induced local MBP translation, as did stimulation in the presence of NMDA or mGluR receptor antagonists or suppressing Fyn kinase with siRNA. AMPA receptor and P2 receptor antagonists were without significant effect (*P < 0.005; **P < 0.001; n = 32 cells for each condition, one cell sampled per dish). (E) MBP mobility was monitored using a photoactivatible GFP–MBP (fig. S12). The mobility of newly synthesized MBP was increased by blocking vesicular release from axons, but not by blocking P2 receptors with suramin (P < 0.001; n = 17 cells from 17 dishes for each condition).