On laminins and laminin receptors and their roles in regeneration and myelination of the peripheral nerve

Abstract

One important prerequisite for successful axon regeneration is that appropriate extracellular molecules are available for outgrowing axons and that receptors for such molecules are found in the regenerating neuron. Laminin associated integrin subunits alpha3, alpha6, alpha7 and beta1 mRNAs are here shown to be present in rat motoneurons, and in the case of integrin subunits alpha7 and gamma1, also immunohistochemical (IHC) labelling of the proteins was demonstrated. Additionally, the expression of integrins in dorsal root ganglion (DRG) neurons was shown. A few DRG neurons expressed integrin subunit alpha6, while integrin subunits alpha7 and foremost beta1 were expressed in a majority of the neurons.

During postnatal development there was a distinct shift in the integrin composition in motoneurons from a stronger expression of the alpha6 subunit to a very clear dominance of alpha7 in the adult. Sciatic nerve transection (SNT) and ventral root avulsion (VRA) in the adult rat induced large upregulations of alpha6, alpha7 and beta1 subunit mRNAs.

Neonatal SNT had much smaller effect on the expression of the studied subunits. SNT of DRG neurons also induced an upregulation of integrin subunits alpha6, alpha7 and beta1 mRNAs in a majority of the neurons. In contrast, integrin mRNA levels were not affected in DRG neurons after dorsal root transection (DRT), where the centrally projecting axons of DRG neurons were injured. By anterograde tracing, immunoreactivity for all integrin subunits learned to be upregulated in the neuron soma was also found in association with growing axons after a sciatic nerve crush lesion (SNC) in vivo. These results suggest that an important part of the response to axotomy of motoneurons is to upregulate receptors for extracellular matrix molecules, like laminins.

Although many studies have shown laminin expression in the sciatic nerve, the developmental regulation and exact composition of the heterotrimers have not been revealed. Laminin alpha2, alpha4, beta1, beta2 and 71 chain rnRNAs are here shown to be expressed at high levels in newborn rat sciatic nerves with declining levels during later developmental stages.

After SNT and SNC, laminin chains alpha2, alpha4, beta1, and gamma1 mRNA levels were upregulated at the site of the injury. Immunohistochemistry of adult human normal and transected peripheral nerves stained positive for laminin alpha2, alpha4, Of and 71 chains in close relation to neurofilament labelled axons, indicating the presence of laminin-2 and -8 in association with axons. These results suggest that both laminin-2 (alpha2beta1gamma1) and laminin-8 (alpha4beta1gammal) are important for the postnatal nerve development and axonal regeneration.

Loss or mutations of laminin alpha2 is known to be the cause in merosin (laminin-2) deficient congenital muscle dystrophy, an inherited disease in which the peripheral nerves display severe dysmyelination, lack of basal lamina and axon degeneration. Here we show that absence of the laminin alpha4chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting and impaired myelination, while the axonal regenerative capacity does not seem to be influenced. The morphological defects are accompanied with signs of ataxia and proprioceptive disturbances.

Further we show that Schwann cells in vitro utilize the integrin receptor alpha6beta1 I in their interaction with laminin-8, but that one or more other integrin PI receptor(s) are involved in the interaction with laminin-2. Also, axonal extension from motoneurons cultured in vitro on different laminin isoforms showed a more permissive role for laminin-2 compared to laminin-8. Thus, we suggest that laminin-2 and -8 have different critical functions in peripheral nerves, mediated by different integrin receptors. Our results also strongly propose that Schwann cells interact with laminin-8 during radial sorting and myelination using the integrin alpha6beta1 I receptor.

In summary this thesis provides evidence for important roles of integrins alpha6, alpha7 and beta1, as well as laminin chains alpha2, alpha4, beta1 and gamma1 during axonal growth and myelination. More specifically, the integrin dimer alpha6beta1, may be linked to laminin-8 (alpha4beta1gamma1) and the integrin dimer alpha7beta1 to laminin-2 (alpha2beta1gamma1) in the peripheral nerve. The temporal regulation of these molecules as well as their effects on neurons and Schwann cells are likely to have implications for peripheral nerve surgery and the study of inherited peripheral neuropathies.