Functional analysis of the CHL1 - hsc70 interaction

co-VI. Discussion

localization was only seen after co-capping of CHL1. This assumption was further investigated by biochemical analysis performing a co-immunoprecipitation of CHL1 and hsc70 from membrane fractions which were derived from either 5-day old or 3-weeks old mice. It was shown that the co-precipitation using crude membranes was much more pronounced in older animals when compared to younger animals although the expression levels of CHL1 were remarkably higher in the 5-day old mice. The crude membranes were further separated in detergent-soluble and insoluble fractions and the subsequent immunoprecipitation of CHL1 revealed a co-precipitation of hsc70 only in the detergent-insoluble fraction that was prepared from 3-weeks old animals. The co-immunoprecipitation using membrane fractions derived from animals of different ages confirmed the assumption that not only the conformation of the binding partners but also the developmental stage of the animals facilitate the interaction of CHL1 and hsc70. As mentioned before, hsc70 shows an elevated expression during early neuronal development, whereas CHL1 becomes weakly detectable at embryonic day 13 and shows the highest expression levels at rather late developmental stages from embryonic day 18 to postnatal day 7 (Hillenbrand et al., 1999).

Furthermore, the biochemical analysis of the CHL1 – hsc70 binding that was carried out with regard to the age of the animals indicated that the interaction between both proteins was more pronounced in older animals than in 5-day old animals. Taken these observations together, a hsc70 function during early developmental stages in mice without an involvement of CHL1 is assumed whereas a functional interaction of CHL1 and hsc70 is facilitated at later developmental stages including the postnatal development in up to 3-weeks old animals and probably also in adults. Thereby, a possible function of the CHL1 – hsc70 interaction is proposed suggesting that the binding of both proteins at later developmental stages may confers stability and/or maintainance of cellular structures to neurons whereas during early development of the mouse brain, hsc70 is probably more involved in alterations of structural elements including conformational changes of cytoskeletal components.

VI. Discussion

reorganization processes of the cytoskeleton following CHL1-dependent neurite outgrowth.

Primary hippocampal neurons derived from CHL1-deficient mice were transfected with either wildtype CHL1 or with CHL1 mutants which were deleted in the hsc70 binding site and neurite outgrowth of transfected cells was stimulated using the anti CHL1 antibody. Although no significant differences in CHL1-dependent neurite outgrowth after transfection of either wildtype or mutant CHL1 were observed in this cellular system, a participation of the interaction between CHL1 and hsc70 in structural alterations of the cytoskeleton can not be excluded. The experimental approach used in this work, namely the transfection of primary neurons using certain CHL1 constructs followed by CHL1-dependent neurite outgrowth, emerged to be a harmful stressor of the transfected neurons. Transfection of CHL1 constructs resulted in massive morphological changes of the cells including a round appearance and the lack of extended neurites that makes a functional analysis of the CHL1 – hsc70 interaction with regard to structural reorganization processes of the cytoskeleton impossible. The question whether the CHL1 – hsc70 interaction is involved in alterations of structure and composition of cytoskeletal elements is still open since this approach could not elucidate the requirement of a functional binding between CHL1 and hsc70 for the promotion of CHL1-dependent neurite outgrowth in primary hippocampal neurons.

As mentioned before, a putative role of the CHL1 – hsc70 interaction was assumed according to the observation that the interaction of both proteins is more facilitated with later developmental stages of the animals and that the localization of the binding is the more pronounced in detergent-insoluble cellular subdomains the older the animals are. Due to this results a functional role of the CHL1 – hsc70 binding was proposed that emphasized an involvement of the interaction in generating stability of intracellular structures. Further evidence that the maintainance of a neuron might be supported by the intracellular interaction of the transmembrane CHL1 with cytoskeletal elements mediated by the heat shock cognate 70 was provided since the interaction between L1 and the 440-kDa-isoform of ankyrinB (ankB) implicates such a function. L1 and ankB are co-localized in premyelinated axon tracts of the developing nervous system, a concomitant down-regulation of both proteins is revealed after myelination and ankB knock-out mice have reduced L1 expression levels in premyelinated axons of long fiber tracts. A functional importance of L1 and ankyrinB in these particular structures is strongly supposed. In fact, analysis of the ankB deficient mice revealed that the optic nerve fibers become dilated and degenerate by day 20 suggesting that the

VI. Discussion

ankyrinB – L1 interaction is essential for the maintainance of premyelinated axons in vivo (Scotland et al., 1998). Unfortunately, the hsc70 knock-out mouse is not available so far such that an involvement of the CHL1 – hsc70 interaction to the maintainance of cellular structures or the degradation of particular neurons is not accessible.

The overlapping distribution pattern of CHL1 and hsc70 in membrane subfractions which were derived from an enriched synaptosomal preparation provide further evidence for a putative functional role of the CHL1 – hsc70 interaction in synapses. In 7-day old animals and in adults, both proteins were present in a so-called raft fraction that was separated from the detergent-insoluble subfraction of synaptosomal membranes. Only in younger animals, a raft localization was identified that solely implicated further interactions to extracellular matrix and/or cytoskeletal structures whereas in adults CHL1 and hsc70 were detectable in rafts with but also without such interactions. Thereby, different functions were suggested for CHL1 and hsc70 in synapses according to the age of the animal. In younger animals, a linkage to structural elements like ECM proteins or cytoskeleton components was more pronounced for CHL1 and hsc70 whereas in adults both proteins implicate a further function that did not involve the interactions with such structural elements. The contribution of hsc70 to presynaptic complexes which are modulatory involved in the neurotransmitter release and the recycling of synaptic vescicles indicates that the interaction between CHL1 and hsc70 may participate in the generation or maintenance of a functional synapse.