Melatonin signalling in Schwann cells during neuroregeneration

Abstract

It has widely been thought that in the process of nerve regeneration Schwann cells populate the injury site with myelinating, non-myelinating, phagocytic, repair, and mesenchyme-like phenotypes. It is now clear that the Schwann cells modify their shape and basal lamina as to accommodate re-growing axons, at the same time clear myelin debris generated upon injury, and regulate expression of extracellular matrix proteins at and around the lesion site. Such a remarkable plasticity may follow an intrinsic functional rhythm or a systemic circadian clock matching the demands of accurate timing and precision of signalling cascades in the regenerating nervous system. Schwann cells react to changes in the external circadian clock clues and to the Zeitgeber hormone melatonin by altering their plasticity. This raises the question of whether melatonin regulates Schwann cell activity during neurorepair and if circadian control and rhythmicity of Schwann cell functions are vital aspects of neuroregeneration. Here, we have focused on different schools of thought and emerging concepts of melatonin-mediated signalling in Schwann cells underlying peripheral nerve regeneration and discuss circadian rhythmicity as a possible component of neurorepair.

Keywords: Schwann cells; basal lamina; circadian rhythm; extracellular matrix reorganisation; melatonin receptors; peripheral nerve injury.

FIGURE 1

Changes in myelin fibre profiles of the regenerating murine femoral nerve after transection. (A) Left: a transversal profile of an intact femoral nerve. Arrows indicate the perineural sheath. Middle: a transversal profile of the proximal stump of the regenerating femoral nerve. Note the thick perineurium (arrows), the enhanced angiogenesis and the degeneration of nervi nervorum after transection. Right: the distal stump of the transected femoral nerve. Scale bar, 100 µm. (B) Left: the intact femoral nerve is composed of homogeneously distributed myelinated and non–myelinated fibres. Middle: sprouting in myelinated (myelinating Schwann cells, mSC) and non–myelinated (non–myelinating Schwann cells, nmSC) areas in the regenerating proximal nerve stump. The thick perineurium (pn) and newly formed blood vessels (asterisks) are visible. Right: distally to the lesion site, myelin decomposition occurs in variable forms within the Schwann cells. Scale bar, 30 µm. Staining: toluidine blue. Specimen: 75–nm–thin sections of resin–embedded non-injured nerves versus injured nerves 10 days after transection in C57Bl6/J mice.

FIGURE 2

Schwann cells at work—ultrastructural patterns of myelin decomposition in the distal stump of a transected murine femoral nerve. (A) A degenerating axon amidst compacted myelin within the Schwann cell cytoplasm. Multiple proteolytic vesicles are visible in the cytoplasmic rim. A basal lamina (arrows) and collagen (co) isolate the Schwann cell from the extracellular matrix. (B) Ovoid figures of partially degraded myelin and peripheral vesicular fusion at multiple sites. The nucleus (nu) of the Schwann cell is visible. (C) A multitude of cytoplasmic vesicles and a few compartments of degraded myelin. The basal lamina (arrows) is an important border, which isolates the dynamic cell interior from the surrounding. (D) Decomposition of a huge mass of myelin within a myelinating Schwann cell. (E) Portioning and proteolytic degradation of myelin (concentric myelin lamellae are visible). (F) Star–like appearance of the basal lamina (arrows) harbouring the processes of the Schwann cells in the cross–section, also known as “Büngner’s band.” This structure is able to accommodate and guide re–growing axons; nu—nucleus. Staining: osmium tetroxide, potassium (III) hexacyanoferrate and post–contrasting with lead nitrate and uranyl acetate. Specimen: 55–nm–thin sections of resin–embedded distal nerve stumps, 10 days after transection performed in C57Bl6/J mice. Scale bar, 2 µm.

FIGURE 3

Ultrastructure of non-myelinated fibre bundles in a non–injured nerve and in a regenerating proximal nerve stump. (A) Multiple non–myelinated axons (ax) hosted by a non–myelinating Schwann cell. Each axon is well separated from the others (B) and more than 40 axons can form a so–called “Remak bundle” (C); nu—nucleus. (D) A single non-myelinating Schwann cell uses its lamellipodia to accomodate axonal sprouts of different calibres (asterisks), but no myelination occurs. Note the difference between the Schwann cell basal lamina of the intact (B) and an injured nerve (E), arrows indicate the electron-dense basal lamina. (F) More than 50 irregularly shaped axonal sprouts (asterisks) can be tightly packed within a non–myelinating Schwann cell. The Schwann cell pseudopodia appear electron–denser than sprouts. Staining: osmium tetroxide, potassium (III) hexacyanoferrate and post–contrasting with lead nitrate and uranyl acetate. Specimen: 55–nm–thin sections of resin–embedded proximal nerve stumps, 10 days after transection performed in C57Bl6/J mice. Scale bar, 500 nm.

FIGURE 4

Schwann cells multitasking at the proximal stump of the injured femoral nerve. (A) In the field of view: four myelinating Schwann harbouring well–preserved myelin lamellae around the injured axons (ax) and at the same time accommodating axonal sprouts (asterisks). (B) The newly formed myelin around the injured axons can vary in its compactness (arrows indicate densely formed lamellae); co—collagen; nu—nucleus. (C) Hyperplastic cells: a giant non-myelinating Schwann cells abundant in sprouts (asterisks) adjoining a myelinating Schwann cell. Note that the myelinating Schwann cell contains two separated myelinated axons (one of a remarkable irregularity), a few cytoplasmic sprouts (asterisks) and proteolytic vesicles (arrows). (D) While re–shaping the myelin structure and compactness (arrows) around the injured axon (ax), a myelinating Schwann cell can concomitantly host more than 10 axonal sprouts (asterisks). (E) A Schwann cell shedding myelin (arrows) while still preserving the axon (ax) and at the same time carrying a sprout (asterisk). Note the abundancy of mitochondria within the axon. In the vicinity: another myelinating and two non-myelinating Schwann cells; nu—nucleus. (F) Hyperplasticity in confined space: arrows indicate densely formed myelin lamellae; asterisks highlight sprouts; arrows indicate mylein shedding. Staining: osmium tetroxide, potassium (III) hexacyanoferrate and post-contrasting with lead nitrate and uranyl acetate. Specimen: 55–nm–thin sections of resin–embedded proximal nerve stumps, 10 days after transection performed in C57Bl6/J mice. Scale bar, 1 µm.

FIGURE 5

Interplay between Schwann cell plasticity and circadian clock during nerve regeneration. The Schwann cells undergo a remarkable transformation during nerve regeneration upon injury. Variations in light’s intensity due to circadian changes or disruption result in an altered activity of the suprachiasmatic nucles and melatonin concentration levels, to which the Schwann cells can respond selectively via their melatonin receptors (MT1/2). Degradation of melatonin by CYP1A2 or changes in blood–nerve barrier’s permeability are regulatory mechanisms allowing a certain amount of melatonin to penetrate the regenerating nervous tissue and to reach the Schwann cells within. MT1/2 may play a role as “chronosensors” to melatonin changes and induce internal clock changes, which allow the Schwann cells to unfold a complex morphology, where multiple events (sprouting, myelin re–assembling, proteolysis, depletion, degradation, and sorting) occur simmulatneously.

FIGURE 6

Melatonin signalling in Schwann cells. MT1 is the dominant receptor mediating melatonin signalling in Schwann cells via three coupled G–proteins—Gi, Gs, and Gq, and also the Ras/Raf/MEK/ERK cascade. Activation of Gs leads to a conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) by the adenylyl cyclase (AC). In turn, cAMP activates protein kinase A (PKA) to phosphorylate mitogen activated protein kinase (MEK) and Raf. MEK activation via Gs occurs through sequential phosphorylation of multiple kinases—beginning with cAMP, followed by PKA, phosphatidylinositol–3–kinase (PI3K), phosphoinositide–dependent kinase 1 (PDK1), PKC, and MEK. Activation of Gs inhibits the adenylyl cyclase and the corresponding downstream signalling. Gq, which is coupled to the MT1 receptor, activates phospholipase c (PLC) and hydrolyses membrane–located phosphatidylinositol–4,5–biphosphate (PIP2) to diacylglycerol (DAG) and inositol–1,4,5–triphosphate (IP3). IP3 activates a calcium channel in the endoplasmic reticulum and induces calcium release into the cytosol. DAG and calcium ions synergistically activate protein kinase C (PKC), which can regulate also MEK. In summary, MEK seems to be the central hub of the melatonin–mediated signalling, where all regulatory pathways converge. However, the upstream kinase of the MAPK cascade Ras becomes also activated by the MT1 receptor (the mechanism is not fully clear). Melatonin upregulates SOX2 (Schwann cell de–differentiation), SHH and Gli1 (signalling components of the Sonic hedgehog pathway mediating Schwann cell migration), as well as BDNF and GDNF (brain and glial derived neurotrophic factors supporting the axo–glial interaction). Melatonin elevates the expression of parkin, which mediates mitophagy in Schwann cells, thus reducing the amount of reactive species as well as oxidative stress. Collagen and chondroitin sulfate proteoglycans, which are components of the extracellular matrix, are being downregulated, while the focal adhesion kinase (FAK) that regulates the interplay between a Schwann cell and its basal lamina is upregulated.