Inhibition of ADAMs proteases stimulates myelination in vitro u

We have shown that BACE1 is required to fully activate the myelination promoting function of NRG1. However, the potential of overexpressed NRG1 to promote myelination remains, albeit at lover levels, even in the absence of BACE1. Therefore, we wanted to investigate possible processing of NRG1 by other proteases in the absence of BACE1. Members of the ADAMs family, ADAM17 and ADAM19, which have been shown to mediate NRG1 cleavage in vitro (Horiuchi et al., 2005; Shirakabe et al., 2001) and are attractive candidates. Unexpectedly, inhibition of ADAMs activity in DRG-Sc co-cultures by a broad spectrum inhibitor (GM6001) promoted myelination and increased the number of myelin segments by twofold in comparison to control culture (Fig. 19). We speculate that this effect might also be observed in vivo as DRG co-cultures replicate other in vivo effects. For instance, BACE1 inhibition almost completely abolished myelination, and viral overexpression of GIEF and HANI-variants in DRG neurons promoted myelination, very similar to finding in transgenic mice (Fig.

18,Fig. 19). The molecular targets and mechanisms responsible for this effect are

currently not clear. However, we found that the association of Sc with the DRG axons is largely unchanged upon treatment with ADAMs and BACE1 inhibitors. This would suggest that proteolytic processing is not required for the initial stage of Sc-axon association but controls axo-glial signalling after an initial contact has been established (Fig. 19). Nevertheless, PI3K and MAPK activities were not significantly changed upon inhibitor treatment (Fig. 19). However, it is possible that other downstream signalling cascades involved in Sc differentiation are affected by the treatment such as calcineurin/NFAT, shown to be activated in Sc after addition of NRG1 in vitro (Kao et al., 2009). Even though the ADAMs inhibitor is not specific for ADAM17, it blocks ADAM17 activity in low-nanomolar concentrations (Moss and Rasmussen, 2007) Based on the importance of NRG1 signalling in PNS myelination (Michailov et al., 2004), one obvious explanation is therefore that inhibition of ADAM17 activates NRG1 to promote myelination. This hypothesis implicates that proteolytic processing can also negatively regulate NRG1 signalling, which is beyond the simple model of proteolysis mediated activation. APP can be cleaved by BACE1 and ADAM17 to produce two functionally different protein fragments (Turner et al., 2003). Therefore, it is possible that a similar scenario exists for NRG1 processing. While currently, no data directly connect ADAMs inhibition with NRG1 processing it will be interesting to study if the GM6001 inhibitor can promote myelination or remyelination after nerve crush in vivo.

This would open and avenue for the therapy of peripheral neuropathies. Interestingly, we observed a similar effect of ADAMs inhibition in co-cultures of CNS neurons and oligodendrocytes. Here the number of myelinating oligodendrocytes was increased by twofold compared to control cultures (Fig. 20). It will be an important task for future experiments to determine if the effect is a consequence of induced oligodendrocytes proliferation at if ADAMs inhibition mainly affects post mitotic oligodendrocytes. It is tempting to speculate that treatment with ADAMs inhibitor could be used in future as a potential treatment to stimulate OPC differentiation in MS lesions and repair of damaged myelin sheaths.

6 S

NRG1 type III-β1 is a master regulator of peripheral myelination (Michailov et al., 2004;

Taveggia et al., 2005). It has been suggested that NRG1 isoforms undergo proteolytic cleavage to generate mature signalling ligands (Mei and Xiong, 2008). Although NRG1 processing by the ADAMs family of metalloproteases has been widely studied, (Horiuchi et al., 2005) the identity of the protease activating NRG1 type III and the exact structure of signaling fragment that regulates myelination remained elusive.

Recently studies of BACE1-/- mutants revealed hypomyelination of the PNS axons and described a physiological role of BACE1 in the PNS myelination (Willem et al., 2006).

Even though processing of the type III NRG1 isoform by BACE1 has been shown in vitro (Hu et al., 2008) the importance of BACE1 processing for NRG1 type III-β1

mediated myelination in vivo has not been directly demonstrated.

Neuronal overexpression of a NRG1 variant mimicking BACE1 processed NRG1 type III-β1 in transgenic mice promotes myelination in the PNS similar to the full length NRG1 protein. This result strongly suggests that BACE1 processing occurs in vivo to activate NRG1 type III-β1 in myelination. Thereby we also showed that back signalling by cytoplasmic tail of NRG1 type III-β₁ is not required for the myelination promoting function against previous speculations. Our finding also implies different signalling function of type III-β₁ and type III-β₃ NRG1 isoforms.

We could show that BACE1 cleavage in the stalk region functionally activate NRG1.

Analysis of the processing pattern of NRG1 type III-β₁ in vivo and in vitro support this first opening cut. In vitro analysis shows no evidence that BACE1 further cuts NRG1 to release EGF domain, but our results strongly suggest cleavage by another protease.

Analysis of the myelination as well as biochemical analysis of mice with GIEF overexpression in the absence of BACE1 will resolve whether second cleavage step by BACE1 occurs in vivo and whether it is necessary for the function in myelination.

BACE1 overexpression does not further promotes myelination in NRG1 type III-β1

overexpressing mice. In the absence of BACE1, overexpressed NRG1 type III-β₁ retains the potential to promote myelination, albeit to the less extent showing for the first time functional interaction between BACE1 and NRG1 in vivo. It remains to be elucidated if BACE1 activity is compensated by another protease or NRG1 function in myelination can be independent of processing mediated activation.

NRG1 protein fragments localize to the cell surface of spinal cord motor neurons and the transport into axonal compartment is limited. Proteolytic processing represents possible rate limiting step for the axonal transport.

Inhibition of the ADAMs proteases promotes myelination in vitro. This finding presents potential approach to stimulate myelination in demyelinating diseases. Whether NRG1 participates in the effect observed with ADAMs inhibitor is not clear, but could be possible mechanism of proteolytic inactivation of NRG1.

7 A

Despite the fact that the following project was discontinued, I will briefly introduce and discus it in the following chapter. I have attempted to generate a NRG1-antibody fusion protein, as an experimental therapy in a mouse model of Multiple sclerosis (MS). This project, done in collaboration with Dr. Moses Rodriguez at Mayo clinic USA, led to the identification of an antibody that following intraperitoneal injection (i.p.) binds to axons in the spinal cord of Theiler’s murine encephalomyelitis virus (TMEV) mediated mouse model of MS.