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One of the quintessential questions in developmental neurobiology is how an undifferentiated neuroepithelium in the embryo can produce unparalleled cellular diversity and specialization in the mature nervous system. One of the most important, yet underestimated types of cells present in the embryonic neural system is the glial cells. Glial cells, which make up an approximate of 90% of the brain (Koob, 2009), are cells present in the central nervous system (CNS) and the peripheral nervous system (PNS), providing support to the brain's neurons and also maintaining homeostasis. Of most primary use, the oligodendrocytes and most Schwann cells produce myelin sheaths (Noback and Strominger, 2005).
This essay will look into the differentiation of two types of glial cells; Schwann cells (PNS) and oligodendrocytes (CNS). Glial specification in structure and function is of utmost importance in the ever increasing neural complexity. The Schwann cells (SC) and oligodendrocytes are cells which arise from completely different lineages (Kandel, 2000). The former develop from the neural crest cells originating from the dorsal tips of the neural tube (Jessen & Mirsky, 1997). Conversely, the oligodendrocytes, develop from the sub-ventricular regions of the lateral ventricles of the neural tube (Noback and Strominger, 2005). However, both these cell types require a common family of molecules for their efficient development, proliferation and functioning, namely, laminin.
Laminins are glycoprotein molecules which are predominantly found in basal lamina of the neural system (Durbeej, 2010). Comprising of one of the key members of the extracellular matrix (ECM), laminins consist of trimeric molecules containing binding sites for varied receptors, like integrins (Colognato, Charles ffrench-Constant, & Maria Laura Feltri, 2005). This essay concentrates on the process with which laminins regulate cell behaviour of two different types of glial cells, arising from completely distinct cell lineages. It tries to put forward the basic concept that several such molecules may exist in the vertebrate system which may be important for the understanding and hence emergence of future strategies to bring about repair. The papers on which this review is based, Yu et al., 2005 and 2009 and Colognato et al., 2009, explicitly show that Schwann cell differentiation and proliferation, and development of oligodendrocytes from their progenitors, are both affected by the deficiency of laminin, respectively.
Origin of SCs and oligodendrocytes:
The neural crest cells give rise to SC precursors which further differentiate into promyelinating or non-myelinating cells. The SCs which are programmed to myelinate, have the essential function of proliferating and extending cytoplasmic processes into axon bundles. Thus, they establish a one-to-one ratio with the large axons which is termed as axonal sorting (Jessen & Mirsky, 1997). There is a large amount of literature corroborating the concept of SCs requiring the formation of a basal lamina to myelinate (Bunge, 1993). SCs lacking laminin have been recorded to have impaired axon interaction, which in turn leads to their stunted proliferation and differentiation (Colognato, Charles ffrench-Constant, & Maria Laura Feltri, 2005).
The earliest appearance of cells which express oligodendrocyte markers is from the ventral forebrain. The OPCs then undergo extensive migration in the ventral to dorsal direction (Kettenmann, Ransom, 1995). In order to form oligodendrocytes which are capable of producing myelin, the OPC has to first differentiate into a post-mitotic oligodendrocyte which then undergoes morphological changes to produce myelin. These changes are brought into effect by existing cell cycles which in turn are partially regulated by laminin (Buttery & C ffrench-Constant, 1999). As is evident from above, laminin is prevalent in two very discrete processes, and hence is a family of intriguing molecules which may have a potential of bringing about simultaneous repair in the CNS and PNS, in the case of glial cell disorders.
Schwann Cells and Laminin
Fig. 1 The Schwann Cell Lineage.- myelinating cells complete differentiation only when they have bipolar morphology, process extension and proliferation; all three regulated by laminins.The axonal sorting performed by SCs is achieved by several steps, namely proliferation, spindle shape formation and process extension. In the paper of Yu et al. (2009), it was exhibited that laminin is required for SC bipolar morphology and process extension. Moreover, SCs lacking laminins do not myelinate at all. Feltri et al. (2002) have also reported that SCs devoid of Î² 1 integrin, a laminin receptor, show incomplete axonal sorting. SCs that lack the laminin subunit usually result in the complete loss of laminin expression due to which there is severe reduction in proliferation (Yu, Chen, North, & Strickland, 2009).
The first aspect Yu et al. (2009) investigated was whether laminins regulate the bipolar morphology of SCs. In order to do this, they used time-lapse live-cell imaging on mouse SC-dorsal root ganglion (DRG) co-cultures as control and mutant mice Cre:fLAMÎ³1 co-cultures, both with the addition on ascorbate to stimulate basal lamina deposition. They reported that after 8 days, most SCs in the control co-culture had bipolar morphologies and also segments of myelin. On the other hand, SCs devoid of laminin did not myelinate and also did not form a bipolar shape. By detecting SCs and myelin using anti-S 100 and anti-MBP (myelin basic protein) antibody respectively and viewing via confocal microscopy, Yu et al. (2009) also showed that SCs which lacked laminins were not able to innervate axon bundles. By means of this procedure, it was clearly observed that the mutant SCs showed a lesser degree of process extension as compared to the control. This suggested that laminins regulate process extension in SCs during sorting.
Rac 1 and Cdc-42 are important GTPase members which also play pivotal roles in axonal sorting. Rac 1 acts downstream of Î² 1 integrin activation (Benninger et al., 2007). A major breakthrough in this paper was the finding of the regulation of both Rac 1 and Cdc-42 by laminins. Rac 1 brings about process extension (Nodari et al., 2007) while activated Cdc-42 is required for cell proliferation. Previously it was reported that Cdc- 42 was activated only by NRG-1 (Grove et al., 2007), which in turn phosphorylated the ErbB receptors. However, this was investigated further by Yu et al (2009), by performing immunoblot assays of the lysates of the co-cultures for phospho-ErbB2. Surprisingly, it was found that the level of phospho-ErbB2 was decreased by a big margin in the co-cultures which lacked laminins. Thus, this implies that laminins regulate both the Cdc42 and Rac 1 pathways. This in turn suggests that laminins regulate the proliferation as well as process extension of SCs. Other findings by the same authors also report the depletion of all laminins once the laminin Î³1 gene is disrupted in the PNS (Yu, M Laura Feltri, Wrabetz, Strickland, & Chen, 2005). This investigation also showed that SCs lacking laminins cannot accomplish the downregulation of Oct-6 transcription factor (which is required for the transition of SCs from premyelinating stages to myelinating SCs - Bermingham et al., 1996). Moreover, by studying the sciatic nerves of mutant mice (with disrupted laminin Î³1 gene), they reported that SCs-axon interactions (mediated by laminins) are crucial for Schwann cell proliferation. They put forward a hypothesis stating that axons are a massive source of SCs mitogens. Hence, an impaired axon-SC relationship gave rise to reduced SC proliferation.
Oligodendrocytes and Laminin
Myelination, the primary function of oligodendrocytes, is an example of intricate cellular specialization. Oligodendrocytes produce large amounts of myelin which wrap around mature axons and facilitate saltatory conduction (Buttery & C ffrench-Constant, 1999). In order to develop into myelin-producing cells, oligodendrocytes undergo two distinct steps. The first consists of the differentiation of oligodendrocyte progenitor cells (OPCs) into post-mitotic oligodendrocytes which express markers for differentiation (like, MBP). The second step consists of a drastic change in the morphology of the oligodendrocytes which is associated with the production of myelin membrane (Buttery & C ffrench-Constant, 1999). It is this membrane which consists of molecules of laminin.
Fig.2 The repression of Fyn results in increase C-terminal Src kinase (Csk) and its binding protein (Cbp). Regulation of signalling pathways is essential for development of OPCs.Colognato et al in 2009 carried out an extensive study on the effect of laminin on oligodendrocyte development involving the Fyn mechanisms. In this paper, they reported that in laminin-deficient mice, the development of oligodendrocytes was delayed. They also found that there was severe accumulation of OPCs in the adult brains.
One of the important signalling molecules in oligodendrocyte development is the Fyn molecule (an Src-family kinase). This regulates the process formation and degree of myelination in oligodendrocytes (Colognato, Charles ffrench-Constant, & Maria Laura Feltri, 2005). In their 2009 paper, they put forward a theory suggesting that the process of oligodendrocyte differentiation is guided by laminin-mediated signalling. By carrying out experiments on mutant mice (dy/dy) with mutations in the LAMA2 gene (for ECM production of laminin-Î±2), they successfully proved the link between laminin and phospho-Fyn and hence oligodendrogenesis (Relucio, Tzvetanova, Ao, Lindquist, & Colognato, 2009). Chun et al in 2003 reported that the corpus callosum and optic nerve were hypomyelinated in the case of dy/dy mutant mice. To evaluate this further, Colognato et al studied both, OPCs as well as oligodendrocytes in dy/dy brains. Their results show that there is significant reduction in the number of mature oligodendrocytes in the mutants when compared with the wild type (wt) littermates. They also investigated this in other regions of the brain and found similar results. On evaluating levels of MBP in the cerebral cortices, they reported that less MBP was observed in the mutants as opposed to the wt.
In order to determine if there is delayed differentiation of OPCs in dy/dy brains, Colognato et al. examined the number of PDGFRÎ±(+) (OPC +) cells in various regions of the brain. They reported a significant increase in PDGFRÎ±(+) cells in the dy/dy mutants than in the wt, proving that there is a delay in differentiation of oligodendrocytes in systems devoid of laminin. An interesting investigation they undertook was to check if laminin promoted OPC maturation. By placing the cultured OPCs in contact with laminin-2 substrates, they reported that the OPC maturation was substantially increased (with increase in phosphorylation of Fyn) as compared to the dy/dy mutant OPCs, thus showing the modulation of Fyn by laminin.
From the above summary of experiments, it is apparent that the work presented by Yu et al., 2005 and 2009 and Colognato et al., 2009 presents strong evidence of the dependency of development of both SCs and oligodendrocytes on laminin. Abnormalities like motor dysfunction, paralysis and neuropathy are present due to an inaccuracy in the development of SCs and oligodendrocytes in the PNS and CNS respectively (Chen & Strickland, 2003). Further research into laminins and their effect on the development and proliferation of these cells could probably help us understand the mechanisms of synthesis and repair failures in greater depth, thus facilitating the evaluation of the role of laminin in the nervous system.
In the case of systems consisting of oligodendrocytes lacking laminins, there may be a factor of increased programmed cell death (Relucio, Tzvetanova, Ao, Lindquist, & Colognato, 2009). However, till date there seems to be no research to support this theory of apoptosis at an earlier time period (the identification of dy/dy mice is not possible at earlier stages). Hence there is a massive scope for studies analysing the laminin gene using knock-out mice. The study of Colognato et al. in 2009 showed for the first time that laminin molecules had regulatory effects on the differentiation of OPCs to oligodendrocytes (by modulating Fyn signalling). There is a major difficulty to detect the presence of laminins in the CNS as basal lamina rich in ECM molecules are rare. Due to this, a massive amount of research has not been put into the effect of laminins on oligodendrocyte development (Colognato, Charles ffrench-Constant, & Maria Laura Feltri, 2005).
Conversely, Yu et al., in 2005 reported that SCs lacking laminin Î³1 expression underwent prenatal apoptosis and hence did not reach full myelinating potential. Moreover, Rosenbaum et al. (1998) reported that the lack of Schwannomin in Schwannoma (SC tumour) cells alters the bipolar shape of SCs, resulting in SCs with rounded morphologies (Rosenbaum et al., 1998). This phenotype has resemblance to SCs lacking laminins. Fascinatingly, rat Schwannoma cells resume their spindle shaped bipolar structure when cultured with laminin (Matsumura et al., 1997). This finding definitely paves a significant application of the effect of laminins on SCs differentiation. In 2005, Yu et al. also hypothesized that the laminin in basal lamina could act as a scaffold for the binding of various growth factors which then influence SCs proliferation. In depth research on this facet of laminins is yet to be done.
Effect of Laminins on:
Degree of myelination
Development of oligodendrocytes from OPCs
Extent of myelination
Schwann cell proliferation
Regulation Effect of Laminins on:
Rac1 and Cdc-42
Future prospects of research on Laminins:
Deficiency of laminins may bring about increase in apoptosis.
Deficiency of laminins may cause prenatal apoptosis.
May unveil a cure for Schwannoma (SC tumour)
Fig. 3 This table summarizes the effect of laminins on both oligodendrocyte and Schwann cell development. The kind of information that laminins may provide certainly seems very intriguing to the mind of a neurobiologist.
The main objective of this essay is to portray the role of laminins in the developmental processes of SCs and oligodendrocytes. Yu et al. and Colognato et al. have tried novel experiments to understand the regulation that laminin can bring about, in the PNS and CNS. They lay a strong foundation for further research in this field which seems to promise finding of high value implications.
In conclusion, it is of extreme importance to understand the functions of ECM molecules like laminin, to serve as a better understanding to the process prevalent in the vertebrate system along with designing strategies to try to enhance repairs. It is strikingly evident from the above findings, that laminins could be holding unexpected roles in the neural system. Needless to state, the laminin family indeed seems to hold a great potential for further research in developmental neurobiology.