Kinesin KIF4A transports integrin β1 in developing axons of cortical neurons
Introduction
Axons grow to their targets during development, and in the mammalian PNS this growth ability is maintained, allowing cut axons to regenerate. In the CNS, however regeneration usually fails, partly due to inhibitory molecules in the CNS environment, partly due to an intrinsic loss of regenerative ability in CNS axons (Chew et al., 2012, Pernet and Schwab, 2012, Bradke et al., 2012). In previous work we have studied the role of integrins in axon growth and regeneration, in the expectation that this family of growth-related molecules would be a key to open up an understanding of how intrinsic regenerative ability is lost as neurons mature (Eva and Fawcett, 2014). Integrins participate in developmental axon growth and in regeneration in the PNS, being found in axon growth cones where they act as receptors for a variety of extracellular matrix glycoproteins (Tomaselli et al., 1993, Gardiner et al., 2005, Blackmore and Letourneau, 2006, Vogelezang et al., 2007, Werner et al., 2000). Integrin expression changes with development, with loss of some integrins that have the potential to promote regeneration (Pinkstaff et al., 1999). For instance, the main integrin ligand in the damaged CNS is Tenascin-C, but the Tenascin-C receptor α9β1 is absent in adult neurons. Expression of α9 integrin in adult DRG neurons enhanced regeneration of sensory axons in the adult spinal cord (Andrews et al., 2009). However inhibitory chondroitin sulphate proteoglycans and NogoA inactivate integrins; this can be counteracted by expression of the integrin activator kindlin, leading to enhanced regeneration of sensory axons in the cord (Tan et al., 2012, Tan et al., 2011, Hu and Strittmatter, 2008).
Integrins can only assist regeneration if they are actually present in the damaged axons, which is not the case in the axons of cortical CNS neurons. Unlike sensory neurons, central neurons become compartmentalized as they mature, with many molecules including integrins being excluded from axons but not dendrites (Bi et al., 2001, Mortillo et al., 2012, Franssen and Zhao, 2014). During early development of cortical neurons, integrins are present in both axonal and dendritic compartments but as maturation progresses integrins disappear from axons. The exclusion from axons of integrins and other molecules necessary for successful regeneration such as ribosomal proteins and trkB may explain the developmental loss of regenerative ability. In this study we have asked whether the kinesin KIF4A transports integrin during development in CNS neurons, and whether changes in its expression are responsible for the loss of integrins in mature axons.
Our focus on KIF4A comes from previous studies, demonstrating that it transports two other molecules with polarised patterns in neurons, and that it is developmentally downregulated (Sekine et al., 1994). KIF4A is involved in the transport of the adhesion molecule L1 in developing hippocampal neurons (Peretti et al., 2000) and in the transport of the ribosomal protein P0 to the tip of axons of developing dorsal root ganglion neurons (Bisbal et al., 2009). The association of KIF4A with L1 occurs in a region with strong homology with FERM (band 4.1, ezrin, radixin, and moesin) domains, which are present on integrins and are involved in integrin signalling via Talin (Peretti et al., 2000).
In the present study, we have demonstrated that KIF4A is developmentally downregulated, associates and co-localises with integrin β1 and that it is involved in integrin transport into immature axons. Interfering with KIF4A by siRNA disrupts integrin transport in young developing axons, however restoration of KIF4A levels in mature neurons triggers apoptosis at high levels, and does not bring integrins back into the axons. KIF4A is therefore necessary for integrin transport in young axons, but other mechanisms prevent integrin entry into mature axons.
Section snippets
Distribution of KIF4A, integrin β1 and α5 in cortical neurons
We investigated the endogenous expression and distribution of KIF4A and integrin β1 in non-neuronal cells and in cortical neurons. In non-neuronal cells, we cultured HEK293, HeLa and undifferentiated PC12 cells and immunostained them against KIF4A. KIF4A positive puncta were predominantly in the nucleus of cells in interphase and at the midzone of cells undergoing mitosis, in agreement with previous findings (Midorikawa et al., 2006) (Fig. 1). In cultures derived from dissociated E18 cortex,
Discussion
Our hypothesis was that KIF4A might be an integrin transporter, and that the disappearance of integrins from mature CNS axons might be due to the developmental downregulation of KIF4A. These ideas were based on previous literature showing that KIF4A transports two molecules with polarised distribution in neurons (L1 and ribosomal protein P0), and that it is developmentally downregulated in neurons and the CNS (Bisbal et al., 2009, Peretti et al., 2000, Sekine et al., 1994). The hypothesis was
Cell culture
Dissociated cortical cultures were established from E18 embryos collected by caesarean-section from a CO2-anaesthetized pregnant Sprague-Dawley rat as described previously in (Marland et al., 2011). Briefly, the embryos were sacrificed by decapitation, placed in ice-cold Ca2 +/Mg2 +-free Hank's balanced salt solution (Gibco 21250) dissecting media and the cortex from both hemispheres were isolated and kept in ice-cold HBSS-CMF. The tissues were then incubated in a solution of HBSS-CMF containing
Acknowledgements
The work in this paper was funded by grants from the Medical Research Council (G1000864), ERC – ECMnuero (294502), The Henry Smith Charity, the EU Marie Curie training programme Axregen, the Christopher and Dana Reeve Foundation, the John and Lucille van Geest Foundation, and the NIHR Cambridge biomedical research centre.
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