Studies of the trafficking and subcellular localization of NRG1 have been hampered by the lack of appropriate antibodies and sensitivity levels of the staining methods. Our transgenic mouse lines that overexpress HA-tagged NRG1-variants offer an opportunity to investigate the subcellular localization of NRG1 in neurons in vivo. By immunostaining with antibodies directed against the N- and C- terminus of NRG1, we were able to show that N- and C- terminal protein products colocalize at the cell surface of spinal cord motor neurons (Fig. 14). WB analysis of spinal cord protein lysates identified a ~70kD N-terminal fragment resulting after processing of the 140kD full length NRG1 (Fig. 17). We suggest that NRG1 is transported to the cell surface to be processed by BACE1. However, processing might already occur in the Golgi
compartment. Thus, even though N- and C-terminal fragments colocalize on the cell surface of transgenic mice that overexpress full length NRG1 type III-β1, it is possible that they are delivered to the membrane already separated by the cleavage in the stalk region. Available BACE1 antibodies did not produce conclusive information about the subcellular localization of BACE1. Therefore, to further elucidate the site of NRG1 cleavage by BACE1 it will be necessary to perform WB analysis after subcellular fractionation, which will allow us to identify if NRG1 is already processed in the intracellular compartment. The GIEF variant is strongly enriched within the intracellular vesicular compartment, but also detected at the cell surface of spinal cord motor neurons (Fig. 14). Endogenous NRG1, detected by the C-terminal antibody is localized in patchy pattern on the neuronal surface which could correspond to lipid rafts micro domains. Interestingly, the endogenous C-terminal fragment occupies distinct membrane microdomains when compared to the GIEF variant. Thus, N- and C-terminal fragments are transported to a distinct membrane compartments. Accordingly, the GIEF variant would than localize to non lipid raft domains. These findings are in line with previously described biochemical assays which revealed that full length NRG1 and the C-terminal fragment harboring the transmembrane domain localize to the lipid raft membrane fraction in transfected cells (Frenzel and Falls, 2001). In contrast, the HA tagged N-terminal fragment was detected in a non lipid raft membrane fraction. In this study lipid raft localization was suggested to be significant for NRG1 signalling. This is in agreement with the proposed function of lipid rafts in the compartmentalization of signaling complexes and formation of platforms for proteolytic processing (Brown and London, 2000; Simons and Ikonen, 1997; Simons and Toomre, 2000). Indeed, in vitro studies showed that the processing of NRG1 by ADAM19 takes place within lipid raft microdomains (Wakatsuki et al., 2004). BACE1 is also compartmentalized to lipid rafts (Riddell et al., 2001) and it is possible that NRG1 type III-β1 processing by BACE1 requires lipid raft colocalization. After processing of the full length NRG1 N-terminal fragment is probably released to the non lipid raft microdomain. We also found that the transport of NRG1 into the axonal compartment is limited. While wt and transgenic variants of NRG1 were prominently expressed in spinal cord motor neurons, sciatic nerve expression was very low or even undetectable (Fig. 14, Fig. 15, Fig. 16, Fig. 17).
Strong overexpression in the GIEF transgenic line increased protein transport into the sciatic nerve above a level that allowed immunodetection. Immunostaining of sciatic nerve cross sections identified an HA-tagged N-terminal fragment on the axonal membrane. Moreover, vesicles containing the N-terminal fragment were present in the axonal lumen presumably transporting the N-terminal fragment from the neuronal cell body. It is still an open question to which extent the C-terminus is transported in to the
axon at all since it was not detected in sciatic nerve of NRG1 overexpressing mice (Fig.
15, Fig. 16, Fig. 17,). In contrast to our findings authors of the study analyzing NRG1 levels in sciatic nerve of wt and Erbin mutant mice detected by WB full length NRG1 protein of 140kD and ~60kD processed C-terminal fragment (Tao et al., 2009). We assume that by increasing in protein levels in the homozygous HANI mice will allow us to resolve this question. Taken together, our findings suggest that the regulated transport of NRG1 into the axon serves as a mechanism to provide appropriate level of NRG1 on the axonal membrane for myelination control compatible with the finding that the amount of NRG1 rather than ErbB receptors appears to be rate limiting for myelin formation (Michailov et al., 2004). Therefore, the amount of NRG1 in the nerve must be tightly regulated to produce the appropriate amounts of myelin. Indeed, our data show that only a fraction of the synthesized NRG1 is transported into the axonal compartment as we detect only small amounts of NRG1 in the sciatic nerve by WB and immunostaining. The identity of the mechanisms involved in regulation of axonal trafficking of NRG1 is currently unknown. We speculate that proteolytic cleavage could be one of the checkpoints. It is also possible that transport into the axon is regulated by the level of endosomal vesicle release. Taken together, we propose that NRG1 accumulates in lipid rafts and it is transported to the neuronal surface to be proteolyticaly cleaved by BACE1. Subsequently, the N-terminal fragment diffuses into non-lipid raft compartments from where it is internalized and transported into the endosome. Vesicles containing NRG1 are subsequently transported from the recycling endosome into the axon and integrated into the axonal membrane. The C-terminal fragment most likely stays behind in the neuronal soma to mediate neuronal survival by back signalling to the nucleus.(Fig. 21).
Fig. 21 Model of NRG1 trafficking in neurons
(1) Synthesized NRG1 and BACE1 transported in vesicles from trans-Golgi network (TGN) to the neuronal cell surface. After proteolytic processing (2) NRG1-N-terminal fragment (green) is localized to distinct membrane compartment (3) to NRG1-C-terminal fragment (red). N-terminal fragment is than internalized (4) and transported to recycling endosome (RE) (5). From RE vesicles containing NRG1-N terminus are transported to the axon (6) and integrated into the membrane. N-terminus on axonal membrane is potentially further cleaved (labeled with question mark) to release the EGF-like domain. C-terminus most likely remains in the neuron soma to mediate back signalling to the nucleus (7).