Regulated intramembrane proteolysis of NRG1 type III dediates postnatal peripheral myelination

Neuregulin-1 (NRG1) type III is a growth factor on the surface of neurons in the peripheral nervous system (PNS). It is required for initial myelination of nerves by Schwann cells after birth and for remyelination after injury. Neuregulin-1 type III is activated by cleavage (shedding) in its extracellular juxtamembrane region generating a membrane-bound N-terminal fragment (NTF) that contains a bioactive epidermal growth factor (EGF)-like domain. This domain signals to neighboring Schwann cells in a contact-dependent manner prompting the cells to initiate myelination. The β-site APP cleaving enzyme 1 (BACE1) was identified as the enzyme that cleaves NRG1 type III and promotes myelination. Consequently, loss of BACE1 cleavage results in dramatically reduced myelin sheaths around nerves in the PNS of BACE1 knockout mice. Besides its role in myelination, BACE1, better known as β-secretase, is also involved in the generation of the neurotoxic amyloid β-peptide (Aβ) which is the main component of amyloid plaques in the brain of patients suffering from Alzheimer’s disease (AD). The Aβ peptide is derived through sequential cleavage of the amyloid precursor protein APP, first by BACE1 in the extracellular domain and subsequently by the γ-secretase in the transmembrane domain (TMD). Inhibition of BACE1 and γ-secretase is therefore considered a promising therapeutic strategy for AD. However, this approach harbors the risk of mechanism-based side effects due to impaired processing of substrates beside APP such as NRG1 type III which is not only a substrate for BACE1 but like APP is also cleaved in its TMD by the γ-secretase. Adding another layer of complexity, ADAM10 and ADAM17, the so-called α-secretases of AD, also cleave NRG1 type III. In the first part of this study, the proteolytic processing of NRG1 type III in its ectodomain was investigated in detail. The precise juxtamembrane shedding sites of BACE1, ADAM10 and ADAM17 were determined by mass spectrometry and two novel cleavage sites of BACE1 and ADAM17 N-terminal of the EGF-like domain were discovered. Cleavage at these novel sites by ADAM17 and BACE1 results in the secretion of the EGF-like domain from NRG1 type III as α-sEGF and β-sEGF, respectively. Using novel monoclonal antibodies generated against the identified cleavage sites the processing of NRG1 type III could also be confirmed in primary neurons. The soluble EGF-like domains were found to be functionally active and induced signaling pathways required for myelination in cultured Schwann cells. Furthermore, β-sEGF rescued the myelination deficit in the PNS of a zebrafish model lacking BACE1, thereby demonstrating its activity in vivo. Using cell culture and the zebrafish model the effects of BACE1- and ADAM17-mediated shedding on the activity of the soluble EGF-like domains were carefully dissected. In contrast to published evidence, however, both the BACE1- as well as the ADAM17-shed sEGF were found to be equally active and to promote myelination in vivo. Together this suggests that NRG1 type III dependent myelination is not only controlled by membrane-retained NRG1 type III but also in a contact-independent manner via proteolytic liberation of the EGF-like domain. The second part of this study investigates the processing of the C-terminal fragment (CTF) which remains after shedding of NRG1 type III. Intramembranous cleavage of the CTF by the γ-secretase was previously shown to release the NRG1 intracellular domain, which acts as transcriptional regulator of proteins involved in neuronal maturation and brain plasticity. Interestingly, a mutation within the TMD of NRG1 type III is associated with an increased risk of schizophrenia linking γ-secretase processing of NRG1 type III to this neurological disorder. Using a novel antibody against the N-terminus of the NRG1 CTF it was possible to detect a NRG1 β-peptide that is secreted during γ-secretase cleavage and could potentially serve as marker for this processing. Moreover, by means of mass spectrometry, the precise cleavage sites within the TMD of NRG1 could be identified. Strikingly, the ɛ-like cleavage site was found to be located exactly at the position of the schizophrenia-associated mutation providing a possible mechanism for the reported interference of this mutation with γ-secretase cleavage. The evidence presented unambiguously establishes NRG1 type III as a γ-secretase substrate and provides a basis for further investigation of the mechanisms which link its processing to the development of schizophrenia. In summary and with regard to BACE1 and γ-secretase being prime targets for a potential AD therapy, the results of this work call for further careful investigation of the consequences of altered NRG1 type III signaling due to chronic treatment with inhibitors.