Research reportLaminins of the adult mammalian CNS; laminin-α2 (merosin M-) chain immunoreactivity is associated with neuronal processes
Introduction
Laminins are large extracellular matrix glycoproteins which are abundantly present in basement membranes of neural and non-neural tissues where they have different distributions 3, 11, 14, 33, 46, 54, 60, 62. Most laminins consist of heterotrimeric assemblies of a longer chain (≈400 kDa) and two shorter chains (≈200 kDa each). Recently, a new nomenclature has been adopted for the various isoforms and combinations of these chains [5]. The longer A and M chains have been renamed α1 and α2, respectively, the shorter B1, S and B2 chains have been renamed β1, β2 and γ1. EHS laminin (α1-β1-γ1) has been renamed laminin-1, merosin is laminin-2 (α2-β1-γ1), S-laminin is laminin-3 (α1-β2-γ1) and S-merosin is laminin-4 (α2-β2-γ1). Characterization of kalinin (also named nicein or epiligrin) and k-laminin have revealed three more genetically different chains (α3, β3 and γ2) which assemble into laminin-5–7 5, 18, 20, 31, 51. Two additional long chains have been cloned more recently (named α4 [26]and α5 [43]). Thus, the number of chain combinations is potentially very large and could be a source of selectivity in cell–cell or cell–matrix interactions. Laminins have various biological activities in vitro, including promotion of neurite outgrowth, cell attachment, cell proliferation and differentiation, and appear to be important for the development, differentiation and regeneration of the nervous system 1, 3, 6, 15, 23, 25, 34, 35, 36, 41, 42, 44, 50, 52, 53.
In the adult PNS, laminin immunoreactivity in Schwann cell basement membranes 4, 38, 39, 40represents only laminin-2 14, 33, consistent with findings that Schwann cells synthesize β1 and γ1 but no α1 chain 10, 45. Neuromuscular junction basement membrane contains laminin-3 24, 54and laminins can bind to dystrophin-associated glycoprotein complex (reviewed in 7, 16) and are involved in neuromuscular attachment and synapse function.
In the adult CNS, laminin immunoreactivity has been described in association with blood vessel basement membranes and reactive astrocytes [38]. We and others 22, 57, 63, 64, 66have reported laminin-like immunoreactivity inside most neurons of the developing and adult rat and human CNS, utilizing polyclonal antisera to laminin-1 (α1-β1-γ1) and monoclonal antibodies to γ1 chain. In fact, mRNA for β1 and γ1 has been revealed by in situ hybridization techniques in retinal ganglion cells [55]and dorsal root ganglion neurons [32]. The role of laminins for CNS neurons is also suggested by the association of laminin-binding proteins with CNS neurons 19, 21, 28, 56, 58.
The present study was aimed at further elucidating the nature of neuronal laminins of the CNS by localizing immunoreactivity with nine specific antibodies against long (α1 and α2) and shorter chains (β1, β2 and γ1) in the adult CNS of various mammals (rat, rabbit, pig and monkey).
Section snippets
Laminin chain-specific antibodies (Table 1)
Mouse monoclonal antibodies 2G9 and 5H2 were made against a reduced 50-kDa fragment of the α2 chain containing the two most C-terminal repeats of the G-domain (G4 and 5). A rabbit polyclonal antibody (PAbl) against a native non-reduced 65-kDa human α2 chain containing the two most C-terminal repeats in the G-domain was affinity-purified on a column of purified human placental laminin-2. Specificity of this polyclonal antibody for basement membrane α2 in brain is described elsewhere [30].
Results
For an overview of the immunocytochemical staining results, see Table 1.
Discussion
Although CNS neurons have been shown to contain laminin-like immunoreactivity (specifically for γ1 chain 22, 57, 63, 66) and mRNA for β1 chain [55], the nature of the neuronal laminin-like molecules is not yet clear. Here, we used nine specific antibodies against various laminin chains to further characterize the presence of laminin molecules in the CNS of four different mammalian species (rat, rabbit, pig and monkey). The main findings are that: (i) laminin-α2 staining is associated with
Acknowledgements
The expert technical assistance of C. Murdock, D. Earwicker and Dr. M. Tian is very much appreciated. We thank Dr. J. Sanes (Washington University, St. Louis, MO) for the antibodies to the S-chain, Drs. M. Paulsson (Köln, Germany) and P. Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ) for the anti-mouse E3 antibody and Dr. L. Eng (Stanford University and VA Hospital, Palo Alto, CA) for the GFAP antibody. We especially thank Drs. J. Johansson (FDA, Laurel, MD) and L. Walker (Johns
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