Polarized membrane assembly is an intricate process, requiring a coordinated synthesis, transport and sorting of proteins and lipids (Figure 6). During last decade, significant advances were made in defining sorting motifs for apical and basal-lateral protein sorting, describing the sorting machinery in the trans-golgi network (TGN) and plasma membrane (PM) of simple polarized cells. MDCK cells have extensively been used for polarized trafficking studies. Another system that underwent an intensive investigation for polarized trafficking, to dendrites and axons, is Neuronal. Trafficking studies of CNS myelin proteins in MDCK cells (Kroepfl and Gardinier, 2001) have yielded valuable insight into how myelin biogenesis might take place. Myelin forming Schwann cells also share some feature of MDCK cells (Figure 6). Oligodendrocytes, compared to both cell types are much more complicated and must possess a unique trafficking and signaling pathway to sort myelin proteins to various myelin growth cones.
Figure 6: Protein sorting and domain organization in polarized cells
A) Postulated mechanisms of post-golgi circuits in MDCK cells (left) and fibroblasts (right). Sorting in the raft circuit (in red) is based on sphingolipid–cholesterol microdomains. Proteins like vesicular integral membrane protein (VIP) and annexins associate and act as stabilizers for other proteins (Fiedler et al., 1994).
Alternatively (in blue), cells employ sorting signals in the cytoplasmic tails and binding at proteins. In the blue circuit NSF–SNAP–SNARE–Rab system is used for vesicular docking and fusion (Fiedler et al., 1995).
B) Schematic organization of the nodal region of myelinating glia (in PNS) is compared to simplified organization of chordate (upper right) and invertebrate epithelial (lower right) cells. The nodal region (red) is masked by Schwann cell microvilli in PNS (astrocytes processes in CNS). The paranodal region is a site of extensive junction formation. It serves as a barrier between the extracellular space at the node and the periaxonal space in the internode. This also separates nodal membrane proteins from the juxtaparanodal proteins. Paranodal loops form extensive autotypic junctions that are radially and circumferentially arrayed:
These include tight junctions (TJ) that provide a presumptive paracellular seal between the periaxonal space and the loops, gap junctions (GJ) that permit direct communication between loops, and adherens junctions (AJ) that promote loop to loop attachment. The apical membrane of epithelia is rendered in red, and the lateral domain, a site of homotypic cell interactions, is rendered in purple. A diffusion barrier between membrane domains is provided by tight junctions in chordates; septate junctions in invertebrates (green), which are orthologous to the paranodal junctions, are interposed between domains. [A and B are adapted from (Simons and Ikonen, 1997) (Salzer, 2003)]
How do the CNS myelin internodes and myelin processes expand? Although, there has been substantial progress in our understanding of the factors that determine glial cell fate, much less is known about the cellular mechanisms that determine how the myelin sheath is extended and stabilized around axons. As Oligodendrocytes enter terminal differentiation and contact neurons, they begin to produce myelin membranes at a remarkable rate (>104 μm2 myelin membrane surface area/cell/day; (Pfeiffer et al., 1993)). During myelination, oligodendrocytes must decide how many times each growing process needs to be wrapped around a segment of an axon. By following biochemical clue displayed by each axon, oligodendrocytes must integrate each signal and respond by delivering proteins and lipids to each growing process, accordingly. These biochemical clues or signals also help oligodendrocytes to discriminate between: glial and neuronal processes, dendrites and axons. Do glial cells rely on biochemical clue, once the myelination is completed, is still a major questions of the field. Understanding how myelinating glia and neurons co-operate to achieve this feat is a challenging and important problem. Current concepts of lipid rafts, which propose the existence of microdomains in membranes, might help to explain how proteins and lipids are delivered to the growing membrane (Figure 6 A). However, such concepts might be less useful for understanding how the myelin macrodomain, with its distinct protein and lipid content, is stably segregated from the plasma membrane of the myelin-forming glial cell. Cholesterol is a major constituent and a rate limiting step in myelination. Transgenic mice with oligodendrocytes that lack an ability to synthesize cholesterol show a delay in myelination that seems to be at least partially compensated by cholesterol uptake (Saher et al., 2005). Lipids are probably targeted to the growing process as a consequence of their interactions with particular proteins (Horvath et al., 1990;
Sankaram et al., 1991). Solving the puzzle about lipid, leads to another puzzle i.e, how proteins are segregated into growing myelin tongue. Most likely this occurs as a result of a combination of factors, such as the specific targeting of proteins during their biosynthesis, cis-association with other proteins and finally by trans-adhesive associations during compaction and axon–glia interaction.
Studies have therefore focused on how oligodendrocytes (OLs) synthesize MBP and PLP and incorporate them into the growing myelin sheath. The discovery that myelin basic protein (MBP) is synthesized in the growing myelin process (Colman et al., 1982; Trapp et al., 1988) on free ribosomes was a major step forwards in understanding of how proteins
might be delivered to the myelin membrane. This was one of the first demonstrations of localized mRNA translation in a eukaryotic cell, and indicated that MBP is incorporated into the growing myelinating process at sites that are quite distant from the oligodendrocyte cell body.
In our study, we have investigated vesicular trafficking to myelin compartment by generating stable oligodendrocyte cell line expressing PLPwt-EGFP. We also transfected primary oligodendrocytes (OLs) to compare trafficking polarization between precursor and mature cells When expressed in cultured OLs, PLP resides in a compartment with characteristics of a late endosome/lysosome (LE/L) compartment. Co-culture with neurons (or cAMP treatment) lead to an increase of PLP on the PM and a disappearance from the LE/L (Trajkovic et al., 2006).
Do neurons give instructions to glial cells? Oligodendrocyte precursor cells (OPCs) in the CNS migrate into developing white matter where they differentiate into postmitotic OLs and produce the myelin sheath. The differentiation of OPCs in terms of changes in gene expression and in morphology has been studied extensively in vitro and in vivo (Pfeiffer et al., 1993). Because OPCs differentiate normally in axon-free culture and express myelin components, a role for neurons was not immediately apparent. OPCs and newly born OLs require astrocyte-derived factors such as PDGF, but OLs become dependent on axonal signals later. Axonal signaling to OLs occurs on at least two levels (Barres and Raff, 1999;
Coman et al., 2005). Electrical activity mediated by extrasynaptic release of adenosine (Stevens et al., 2002) is required for proliferation of OPCs. Additionally, contact-mediated neuronal signals play important roles in OPC and Schwann cell differentiation and myelination (Corfas et al., 2004). Michailov and colleagues have shown that the levels of neuregulin 1 type III overexpression by axons results in hypermyelination in PNS (Michailov et al., 2004). In a follow up study Salzer and colleagues have shown neuregulin 1 type III also determine the ensheathment fate of axons in the PNS (Taveggia et al., 2005).