Rio-Hortega's drawings revisited with fluorescent proteins define a cytoplasm filled channel system of CNS myelin

A century ago this year, Pío del Río-Hortega (1921) coined the term ‘oligodendroglia’ for the ‘interfascicular glia’ with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm-delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm-filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td-tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río-Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the ‘unrolled’ sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under-appreciated complexity of the myelinic channel system in mature myelin sheaths

Biogenesis and maintenance of cytoplasmic domains in myelin of the central nervous system

Myelin is a multi-lamellar membrane structure, produced by oligodendrocytes which are special glial cells, that myelinate axons in the central nervous system (CNS) (Aggarwal, Yurlova, & Simons, 2011; Vassall, Bamm, & Harauz, 2015). The main role of these tightly-packed and stable structures is to electrically insulate the axon. During the biogenesis of myelin, two processes have to be coordinated. At first, the incorporation of myelin adjacent to the axon at the innermost tongue is accompanied by the lateral expansion of newly formed layers. At the same time, a complex system of cytoplasmic channels (CPCs) is formed, enabling membrane trafficking from the cell body to the leading edge in thin-caliber-axons of the immature optic nerve (Snaidero et al., 2014). These channels are known in the peripheral nervous system (PNS) as Schmidt-Lanterman Incisures, but have not been yet established in the CNS (Gould, Byrd, & Barbarese, 1995; Small, Ghabriel, & Allt, 1987). The development of an improved protocol for high-pressure freezing (HPF), allowed us to better preserve the native myelin ultrastructure close to its native state. Using HPF and freeze-substitution for transmission electron microscopy (TEM), we were able to visualize a system of cytoplasmic (myelinic) channels within myelin surrounding large-caliber axons in the CNS for the first time. In line with their presence in developing myelin lamellae, here, we present how a system of interconnected CPCs is organized in mature myelin of axons with different calibers. Beside the morphological analysis of these channels by TEM, we combined different in vivo and in vitro approaches to describe the biogenesis, molecular structures, and possible roles of CPCs. We elucidated a mechanism that regulates the formation and determines the molecular organization and their involved key components. In this study, we identified 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNP) as an essential determinant in generating and maintaining cytoplasmic domains within compact myelin sheaths. Our observations provide evidence that the protein-protein interaction of CNP and filamentous actin (F-actin) results in the formation of a stable structure that helps to keep opposing myelin leaflets separated. The close interaction of CNP and F-actin prevents membrane compaction that is exercised by the classic myelin basic protein (MBP)

OLIGODENDROCYTE 2PHATAL REVEALS DYNAMICS OF MYELIN DEGENERATION AND REPAIR

Oligodendrocytes are responsible for producing myelin in the central nervous system. This lipid-rich coating along axons helps to increase action potential velocity, provide metabolic support to axons, and facilitate fine-tuning of neuronal circuitry. Demyelination and/or myelin dysfunction is widespread in neurodegenerative diseases and aging. Despite this, we know very little about how individual oligodendrocytes, or the myelin sheaths they produce, degenerate. Myelin repair, carried out by resident oligodendrocyte precursor cells (OPCs), is known to occur following myelin damage in certain contexts. We sought to investigate the cellular dynamics of oligodendrocyte degeneration and repair by developing a non-inflammatory demyelination model, combining intravital imaging with a single-cell ablation technique called 2Phatal. Oligodendrocyte 2Phatal activated a stereotyped degeneration cascade which triggered remyelination by local OPCs. Remyelination efficiency was dependent on initial myelin patterns and dynamic imaging revealed rapid repair with near-seamless transitions between myelin loss and remyelination, a process we call synchronous remyelination. A subset of highly branched OPCs executed this remyelination, pointing towards demyelination-associated morphological signatures of fate. Age-related demyelination mirrored the degenerative cascade observed with 2Phatal; however, remyelination in aging was defective due to failed oligodendrogenesis. Thus, oligodendrocyte 2Phatal uncovered novel forms of rapid remyelination that restore myelin patterns in the adult but are absent in aging. We go on to demonstrate that the maturation state of oligodendrocytes determines the dynamics and mechanism of cell death. Premyelinating and newly formed oligodendrocytes degenerate more rapidly than mature oligodendrocytes, but faster than OPCs, following 2Phatal. Furthermore, they appear to utilize a caspase-dependent form of cell death, while mature oligodendrocytes do not. This new insight suggests that different cell death mechanisms are used by these two populations, necessitating distinct strategies to protect preestablished and new oligodendrocytes in human aging and/or disease

Development of techniques for time-lapse imaging of the dynamics of glial-axonal interactions in the central nervous system

Background: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualise the various stages of myelination. Methodology/Principal findings: Two approaches have been taken to follow myelination over time i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. The data demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, evidence from time-lapse fluorescence microscopy and confocal microscopy suggest that the oligodendrocytes’ cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs which then extend longitudinally along the axons. From this model it is predicted that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion: These experiments show the feasibility of visualising the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualisation of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

The axon-myelin unit in development and degenerative disease

Axons are electrically excitable, cable-like neuronal processes that relay information between neurons within the nervous system and between neurons and peripheral target tissues. In the central and peripheral nervous systems, most axons over a critical diameter are enwrapped by myelin, which reduces internodal membrane capacitance and facilitates rapid conduction of electrical impulses. The spirally wrapped myelin sheath, which is an evolutionary specialisation of vertebrates, is produced by oligodendrocytes and Schwann cells; in most mammals myelination occurs during postnatal development and after axons have established connection with their targets. Myelin covers the vast majority of the axonal surface, influencing the axon's physical shape, the localisation of molecules on its membrane and the composition of the extracellular fluid (in the periaxonal space) that immerses it. Moreover, myelinating cells play a fundamental role in axonal support, at least in part by providing metabolic substrates to the underlying axon to fuel its energy requirements. The unique architecture of the myelinated axon, which is crucial to its function as a conduit over long distances, renders it particularly susceptible to injury and confers specific survival and maintenance requirements. In this review we will describe the normal morphology, ultrastructure and function of myelinated axons, and discuss how these change following disease, injury or experimental perturbation, with a particular focus on the role the myelinating cell plays in shaping and supporting the axon

Myelination in the auditory brainstem

The evolution of myelin was a major key event in vertebrates which aimed to enhance conduction velocity of electrical impulses in axons. Distinct myelination patterns along axons can shape the speed and timing of action potentials. Exact arrival time of inputs at target neurons are crucial for proper neural circuit function. Two key determinants for tuning conduction velocity of myelinated axons are the length of individual myelin sheaths together with the axon diameter. However, it remains unanswered who determines specific myelination patterns along axons – the oligodendrocyte or the axon? And further, when and how do structural parameters of myelinated axons develop in neural circuits in general, in terms of their functionally relevant myelination patterns, axonal morphology and nodes of Ranvier? A system with highest temporal demands is the mammalian sound localization system. Globular bushy cell (GBC) axons involved in circuits processing sound location information are some of the fastest and most precise conducting axons in the mammalian central nervous system. In the Mongolian gerbil (Meriones unguiculatus) GBCs that are tuned to low sound frequencies transmit sound signals to the binaural comparator neurons in the medial superior olive (MSO) where the arrival time of sound at the two ears (interaural time differences; ITDs) is computed. These differences can be as low as only a few microseconds and thus, computation of ITDs relies on explicitly fast and highly precise axons. To cope with the need for exact input timing, low-frequency GBC axons exhibit specific structural adaptations to adjust conduction velocity. Their exceptional thick axons combined with comparably short internodes result in unusual low ratios of internode length to axon diameter (L/d ratios) which in turn increase the conduction velocity along their axons. To gain insight into when and how the specific myelin sheath lengths, axon diameter and thus L/d ratios are established, we characterized the developmental time course of these structural parameters at timepoints before and after the onset of hearing. Our findings show the internode length is set prior to a significant axon diameter increase. While the internode length is established already two days before hearing onset, which is at P12, the axon diameter only increases five days after hearing onset, and thereby decreasing its L/d ratio. This strongly suggests that, at least in GBCs, the axon itself is the key determinant in ensuring that the required conduction velocity is met by adjusting its diameter retrospectively. Together with the length of myelin sheaths and the axon diameter, nodes of Ranvier are critical determinants of action potential speed and timing of and therefore the development of all these structures must be tightly regulated. By assessing the development of nodes of Ranvier we found that axon and node morphology by and large mature synchronously. Early nodal clusters appear already when myelination of GBC axons is initiated at P6/P7 and these premature clusters subsequently progress until reaching maturity during the 4th postnatal week. Interestingly, we were able to show that node maturation depends on the location along the axon with nodes closer to the cell body develop earlier compared to nodes close to the synaptic terminal

The Axon-Myelin Unit in Development and Degenerative Disease

Axons are electrically excitable, cable-like neuronal processes that relay information between neurons within the nervous system and between neurons and peripheral target tissues. In the central and peripheral nervous systems, most axons over a critical diameter are enwrapped by myelin, which reduces internodal membrane capacitance and facilitates rapid conduction of electrical impulses. The spirally wrapped myelin sheath, which is an evolutionary specialisation of vertebrates, is produced by oligodendrocytes and Schwann cells; in most mammals myelination occurs during postnatal development and after axons have established connection with their targets. Myelin covers the vast majority of the axonal surface, influencing the axon's physical shape, the localisation of molecules on its membrane and the composition of the extracellular fluid (in the periaxonal space) that immerses it. Moreover, myelinating cells play a fundamental role in axonal support, at least in part by providing metabolic substrates to the underlying axon to fuel its energy requirements. The unique architecture of the myelinated axon, which is crucial to its function as a conduit over long distances, renders it particularly susceptible to injury and confers specific survival and maintenance requirements. In this review we will describe the normal morphology, ultrastructure and function of myelinated axons, and discuss how these change following disease, injury or experimental perturbation, with a particular focus on the role the myelinating cell plays in shaping and supporting the axon

miRNAs AS POTENTIAL REGULATORS OF MYELIN BASIC PROTEIN RECOVERY DURING DEVELOPMENT IN A MURINE MODEL OF PHENYLKETONURIA

Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages. These cerebral alterations are accompanied by myelin basic protein (MBP) impairment, which could be the reason of the clinical traits mentioned above, as MBP is crucial for the correct assembling of the myelin sheath. In this study, we analyzed MBP protein and mRNA expression on brains of WT and phenylketonuric (ENU2) mice during post-natal development (14-60-180-270-360-540 post-natal days, PND). The results showed a progressive MBP protein expression recovery during post-natal development, together with an unaltered MBP mRNA expression. Furthermore, for the same time intervals, a significant decrease of the phenylalanine concentration in the bloodstream of PKU mice was detected, as well as in the PKU mice brains from 14 to 60 PND. To try to explain this scenario, we hypothesized a hindrance during MBP translation in the early development, leading us to perform a microRNA microarray analysis on 60 PND mice. Microarray output and the following in silico analyses underlined the potential role of microRNAs in the PKU cerebral outcomes. In addition, in order to link predictive analysis with concrete data, we performed a proteomic assay on ENU2 brains of 60 and 360 PND. Taken together, we assessed miR-218-1-3p, miR-1231-3p and miR-217-5p as the most promising microRNAs, since that an alteration on their predicted and downregulated targets (MAG, CNTNAP2 and ANLN, respectively) could indirectly lead to a low MBP protein expression. Moreover, their expression shows an opposite trend to that observed for MBP protein during development, except for miR-217-5p. Furthermore, target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and treatment, emphasizing the possible role of differentially expressed microRNAs in PKU brains, especially during early development