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.