In this study, NRIP localized in membrane, nucleus and cytoplasm, including in ER, Golgi apparatus, and cell membrane of HEK293T cells (Fig.1). In Ya-Ju Han’s thesis also showed NRIP localized on C2C12’s cell membrane. Hence, NRIP is a membrane bound protein. Besides, Yun-Hsin’s thesis demonstrated that NRIP colocalized AChR, rapsyn and ACTN2 on muscle cell membrane. AChR components, such as rapsyn and ACTN2, are known to bind directly or indirectly to AChR and induce AChR clustering (Dobbins et al., 2008; Marchand et al., 2002). Previous study of rapsyn demonstrated that rapsyn localizes at Golgi apparatus, transports with AChR to cell membrane through exocytic pathway. The AChRs presented on COS-7 cell surface are decreased when expressing AChR alone compared to co-expressing rapsyn and AChR. Furthermore, rapsyn colocalizes with AChR in post-Golgi vesicles and cell membrane, indicates that rapsyn can escort AChR to membrane and play a role in stabilization of surface AChRs (Marchand et al., 2000; Marchand et al., 2002). AChR’s stability on surface is an equilibrium between rates of insertion and removal of AChRs, and it is regulates by dystrophin-glycoprotein-complex (DGC) (Aittaleb et al., 2017; Bruneau and Akaaboune, 2006). The number/density of AChRs at NMJ is decreased in mice with deficient DGC proteins, such as α-syntrophin, utrophin and α-dystrobrevin (Adams et al., 2000;
Martinez-Pena y Valenzuela et al., 2011; Pawlikowski and Maimone, 2009). Rapsyn has
interaction with DGC proteins, α-syntrophin and α-dystrobrevin, that is mediated by utrophin and maintain AChRs’ stability (Aittaleb et al., 2017). On the contrast, recent study provides evidence that AChR is responsible for rapsyn’s targeting to membrane, while AChR can locate on membrane in rapsyn deficient C2C12 (Chen et al., 2016). As shown on Figure 1, NRIP located in ER, Golgi apparatus and cell membrane, and colocalized with AChR components on cell membrane (Yun Hsin thesis). When NRIP overexpression in C2C12 cells, the cluster formation was enhanced compared with control vector only (Fig. 5B); suggesting that NRIP may involve in cluster formation and be able to escort AChR to cell membrane (Fig.5D). Collectively, NRIP is a membrane-bound protein and colocalizes with AChR complexes in cytosol and cell surface, which is similar to rapsyn.
In this study, we found NRIP as one of an AChR complex components, and participates in AChR clusters formation and neuromuscular junction formation.
Previously, in Szu-Wei Chang and Ssu-Yu Lin thesis unpublished results demonstrated that NRIP could directly bind to ACTN2 EF-hand through IQ domain. Here, we demonstrated that NRIP could bind with AChR complexes including rapsyn, ACTN2 by BTX (a neurotoxic protein that binds to AChR) pull down assay both in C2C12 cells and mouse gastrocnemius muscles (unpublished Hsin-Hsiung Chen’s data, supplementary Fig.2). The rapsyn, AChR and ACTN2 forms AChR complex at postsynaptic muscle
membrane (Wu et al., 2010). Rapsyn, as a AChR complex component, can bind directly to AChR. Rapsyn is the earliest identified AChR-binding protein, which is found by large-scale purification of membrane fragment in AChR in Torpedo at 1970s (Cohen et al., 1972;
Sobel et al., 1977). Rapsyn was then been further studied to induce AChR clustering while co-transfection into non-muscle cells, such as QT-6, COS7, and HEK293T cells, and loss its AChR clustering ability when coiled-coil domain of rapsyn are truncated (Phillips et al., 1991; Ramarao and Cohen, 1998; Yu and Hall, 1994). ACTN2 can bind to rapsyn directly, and associates with AChR clustering at NMJ (Dobbins et al., 2008).
Adenomatous polyposis coli (APC), a tumor suppressor protein, can bind specific to β subunit intracellular loop directly (Wang et al., 2003; Wu et al., 2010). APC mainly localizes in cytoplasm and nucleus in the absence of agrin, while co-aggregates with AChR on C2C12 cell membrane when given neural agrin. Besides, the interaction of APC and AChR β loop is required for agrin induced AChR clustering on C2C12 surface (Wang et al., 2003). As a Wnt signaling molecule, APC may also regulate stability of β-catenin, which regulates AChR clustering via binding with rapsyn and α-catenin (Wu et al., 2010;
Zhang et al., 2007). Taken together, NRIP can interact with AChR complex components, implying that NRIP is a novel structural component of AChR complex.
Here, we illustrated that NRIP could interact with AChR four subunits and the WD40 domain of NRIP is responsible for AChR α binding. NRIP could bind each subunit, while
AChR-δ revealed lower binding ability compared with AChR-α (Fig. 2). AChR is compose of α, β, γ and δ subunits, they assemble into α2βγδ in endoplasmic reticulum (ER) membrane and transport to cell membrane through exocytic pathway (γ is replace to ε in adult human) (Marchand et al., 2002; Wanamaker et al., 2003). For AChR binding, rapsyn is known to anchor AChR α, β and ε subunit’s α helical domain of intracellular loop, while β subunit shows highest clustering and binding ability to rapsyn (Lee et al., 2009). In this study, we demonstrated that NRIP can bind directly to AChR α, β and δ subunits, while δ subunit showed lower binding ability than other subunits (Fig.2). Four subunits of AChR are individual gene products, but share some common structure:
extracellular large NH2-terminal domain; four conserved transmembrane (TM) domain;
one cytoplasmic loop with variable size and amino acid sequence and a extracellular COOH-terminal sequence (Albuquerque et al., 2009; Unwin, 2013)(supplementary Fig.1).
The largest intracellular domain that locates between TM3 and TM4 is composed of a mix of α-helical and β-strand structure (Albuquerque et al., 2009; Unwin, 2005). The variety of intracellular domain of AChR subunits contributes to different protein-protein interaction, including AChR assembly, subcellular localization and stability (Albuquerque et al., 2009; Kracun et al., 2008). In studies of rapsyn’s binding with AChR subunits, rapsyn interacts with subunits’ intracellular loops with different affinities (Lee et al., 2009). The intracellular loop amino acid sequences of 4 subunits showed little
similarity, which form various α-helices on intracellular loop. For AChR α, β subunits, there are α-helices of ~40 amino acid each on intracellular loop, while δ subunit loop is predicted to contain shorter α-helix (Le Novere et al., 1999; Lee et al., 2009). The best binding of rapsyn to AChR is thourgh α-helical domain of β subunit’s intracellular loop, and δ subunit loop with shorter α-helix is unable to bind with rapsyn (Lee et al., 2009).
For another AChR binding protein APC, it only binds to AChR β subunit, indicating the variety of interaction protein between four subunits (Wang et al., 2003). This may explain why NRIP showed different binding ability to AChR subunits, due to the difference of intracellular loop. Besides rapsyn, Lrp4 and MusK are colocalized with AChR on postsynaptic muscle membrane (Valenzuela et al., 1995; Wu et al., 2010). Mice lacking MusK have evenly distributed AChRs and died at birth due to failure of neuromuscular synapse, indicating its importance in NMJ formation (Burden et al., 2018; Lin et al., 2001;
Valenzuela et al., 1995). Phosphorylation of Musk by agrin interacts with various protein, such as Dok-7, Dvl and rapsyn, which participate in MusK activation, AChR clustering and scaffolding (Wu et al., 2010). MuSK and rapsyn can co-cluster in QT-6 cells through its binding to rapsyn via the fourth immunoglobulin-like domain. The fourth immunoglobulin-like domain is also associated with AChR clustering on myotubes, suggesting that the domain required for rapsyn’s binding of MuSK in QT-6 cells is also required for rapsyn-AChR clustering in myotubes (Zhou et al., 1999). In this study, we
add NRIP as one novel AChR-binding protein.
Further we found that NRIP could bind with AChR-α through WD7 domain on C terminal (Fig.4, supplementary Fig.S3) using immunoprecipitation of NRIP mutants and AChR α subunit. NRIP contains seven WD40 domain and one IQ motifs (Chang et al., 2011; Tsai et al., 2005). IQ motif had been reported to bind to calmodulin (CaM) or EF-hand protein (such as ACTN2) (Bhattacharya et al., 2004). WD40 domain is a 40-66 residue sequence units typically contain WD40 dipeptide at C-terminal, is folded into β-propeller architecture in protein (Xu and Min, 2011). WD40 domain has low level of sequence conservation and a diversity of function, tens of WD40 structure are determined so far. WD40 domain proteins are known to function as protein-protein or protein-DNA platform, interacts with various proteins, peptides and nuclei acid (Stirnimann et al., 2010;
Xu and Min, 2011). Like rapsyn, NRIP ‘s binding to AChR through WD7 domain may correlate AChR clustering ability. Similiarly, Coronin 6, a muscle-specific Coronin family member, contains 5 WD40 domain on N-terminal and one coiled-coil domain on C-terminal. Coronin 6 is known to regulates AChR clustering and stabilization by modulating actin cytoskeletal anchorage of AChRs via coiled-coil domain. Although the interaction of Coronin 6 with other AChR components remains unclear, The WD40 domain of Coronin 6 acts as platform for multiple protein interaction, such as rapsyn, and involve in AChR clustering (Chen et al., 2014). This raises a possibility that WD40
repeats’ function as protein-protein interaction adapter is important for protein binding to AChRs.
Here, we demonstrated that NRIP’s binding to AChR-α was correlated with AChR cluster formation in 293T cells. Similarly, the rapsyn’s coiled-coil domain is responsible for AChR binding and clustering in 293T cells (Ramarao et al., 2001). Furthermore, in C2C12 cells, agrin increases rapsyn’s interaction with surface AChR, and the increased rapsyn-AChR interaction correlates with more AChR clustering on cell membrane (Moransard et al., 2003). This reveals that AChR’s binding is associate with AChR clustering formation in cells. Our results revealed the binding and cluster formation between AChR α subunit and NRIP mutants in 293T cells (Fig.5-6); FL, NRIP-ΔIQ and NRIP-C, which showed binding affinity to AChR α subunit in biochemistry assay, could induce AChR cluster formation in 293T cells. C-ΔWD7 lost AChR-α binding ability, therefore had decreased AChR cluster number in 293T cells. This indicates that NRIP’s binding to AChR-α correlates with AChR cluster formation in cells. Although NRIP-N showed weak AChR-α binding ability in biochemistry assay coupled with the decreased AChR cluster number similar to C-ΔWD7 group. This may be due to NRIP-N expressed only in nucleus, not in cytoplasm (Fig.6A) resulted in the failure to form AChR clusters.
In this study, we further confirmed NMJ formation between motor neuron axon and
muscle is correlated with the AChR binding resulted in cluster formation in cell lines.
During NMJ formation, AChR clustering at postsynaptic membrane through agrin-Lrp4-MuSK signaling pathway. Agrin that released from presynaptic motor neuron would phosphorylate MuSK through Lrp4, further triggers signaling pathways that induce stabilization of AChR clusters, like rapsyn for example (DeChiara et al., 1996; Kim et al., 2008). AChR complex component, such as rapsyn, have ability to bind directly with AChR and participates in AChR cluster formation and stabilization in NMJ (Burden et al., 2018). In previous study, we demonstrated that global NRIP knockout (gKO) mice have impaired motor performance and delayed muscle regeneration (Chen et al., 2015).
We further generated muscle specific NRIP knockout (cKO) mice, and examined muscle weakness with abnormal NMJ architecture and axonal denervation at 16 weeks compared with WT (Chen et al., 2018). The abnormal NMJ architecture includes decrease NMJ area and denervation of motor axon terminal at muscle. Besides, there are loss of motor neuron in cKO spinal cord at age 16 weeks (Chen et al., 2018). These indicates that deprivation of NRIP in muscle would affect NMJ formation and stabilization in adult mice. AAV-NRIP-C, which showed AChR binding (Fig. 2) and clustering in cells (Fig. 4), gene therapy in NRIP cKO could increase NMJ area; but AAV-C-ΔWD7, with negative AChR binding (Fig. 2) and clustering in cells (Fig. 4), not effect on restoration of NMJ abnormality of NRIP cKO (Fig.8); hence NMF formation are correlated with AChR
binding and AChR cluster formation. In our study, NRIP is an AChR complex, and its cluster formation in cells is related with NRIP binding with AChR. Furthermore, the binding to AChR through WD7 correlates to AChR cluster formation on postsynaptic muscle membrane in vivo. This further confirm the importance of WD7 domain in NMJ formation, due to its AChR binding ability. Collectively, NRIP interacting AChR can result in cluster formation in cells and NMJ formation in mice.
NMJ formation is important for muscle contraction. During innervation of NMJ, agrin induces phosphorylation of AChR β subunit through phosphorylated MuSK, further recruit additional rapsyn binding with AChR. The recruitment of additional rapsyn also gather more AChR that previously binding with rapsyn to cluster together, induce AChR cluster formation on membrane in C2C12 cells. (Borges et al., 2008; Lee et al., 2009).
These reveal that rapsyn’s binding to AChR is associated with its AChR cluster formation and stabilization on postsynaptic muscle membrane through agrin-Lrp4-MusK signaling pathway (Borges et al., 2008; Brockhausen et al., 2008; Burden et al., 2018). Additionally, rapsyn’s E3 ligase activity in AChR clustering, the rapsyn-induced AChR clustering in 293T cells corresponding to rapsyn-induced AChR clustering on C2C12 cell membrane and NMJ formation in mice (Li et al., 2016). In rapsyn+/- (heterozygote) mice, which express less rapsyn in muscle than WT mice, there are decreased AChR aggregation at postsynaptic muscle membrane (Brockhausen et al., 2008).
Here, we demonstrated that NRIP-C fragment could rescue the NMJ abnormality and motor neuron survival in NRIP cKO mice. AAV gene therapy of AAV-NRIP-C and AAV-C-ΔWD7 to NRIP cKO mice demonstrated that AAV-NRIP-C was able to rescue NMJ formation and motor neuron degeneration, while AAV-C-ΔWD7 wasn’t (Fig.7-9).
For patients of Congenital myasthenic syndromes (CMS) with deficiency of NMJ component, like agrin, Lrp4, MusK and rapsyn, have impaired NMJ development (Engel et al., 2015; Ohno et al., 2002). This reveals the importance of AChR-related component in NMJ formation and maintenance. In Yun-Hsin’s thesis, gene therapy of intramuscular injection of AAV-NRIP can rescue NMJ integrity and motor neuron degeneration in cKO mice. In this study, we compared the efficiency of AAV-NRIP-C and AAV- C-ΔWD7 gene therapy in NMJ formation. AAV-NRIP-C treatment could rescue NMJ area and denervation of motor axon terminal, while AAV-C-ΔWD7 treatment could not. For motor neuron degeneration, AAV-NRIP-C treatment prevented motor neuron death in cKO spinal cord, AAV-C-ΔWD7 did not in contrast. Taken together, AAV-NRIP-C have ability to improve NMJ formation and maintenance of cKO mice at age 16 weeks, but lost it when WD7 domain is truncated. Compare data of AAV-NRIP-C and AAV-C-ΔWD7 to AAV-NRIP, which was done by Yun-Hsin, we could see that AAV-NRIP-C had similar efficiency of rescuing NMJ integrity and motor neuron degeneration (NMJ area: 1 vs.
1.25, P<0.001; denervation fold: 1 vs. 0.58, P<0.05; α-motor neuron: 1 vs. 1.35, P<0.01,
Fig.10). Hence except AAV-NRIP could rescue NMJ formation and motor neuron survival, the AAV-NRIP-C (only containing IQ, WD6 and WD7) had a function for restore NMJ abnormality of NRIP cKO. In the future, AAV-NRIP-C has a potential as a therapeutic drug for the treatment of NMJ impairment, such as ALS, myasthenia gravis (MG) and Congenital myasthenic syndromes (CMS).
As to the role of NRIP on NMJ, NRIP could interact with AChR, ACTN2, and actin (unpublished results); NRIP may act as a scaffolod protein to stablilize NMJ formation.
Like the dystrophin glycoprotein complex (DGC) at neuromuscular junction, that compose of α-syntrophin, α-dystrobrevin, dystrophin utrophin and dystroglycan, is important for structure integrity of synapse and muscle (Blake et al., 2002). α-syntrophin and α-dystrobrevin form complex with rapsyn via utrophin in AChR-rich domain, and contribute to AChR stability at NMJ (Aittaleb et al., 2017). On the other hand, microtubule actin cross linking factor1 (MACF1) colocalized with rapsyn and AChR at NMJ, can recruit microtubule network and maintain AChR density at NMJ (Oury et al., 2019). This reveals the importance of AChR related scaffold protein that support AChR stably clustering on cell surface at NMJ.
In conclusion, NRIP is a membrane-bounded AChR complex component. NRIP can bind to AChR-α reciprocally through WD7 domain on C terminal. With WD7 domain’s binding ability to AChR, NRIP is able to form AChR clusters in 293T cells. Furthermore,
the cluster formation examined in vitro is related to AChR cluster formation at NMJ in vivo. By AAV gene therapy we can see that AAV-NRIP-C treatment is able to improve NMJ integrity and motor neuron survival in NRIP cKO mice, similar to AAV-NRIP treatment, due to WD7 domain’s binding to AChR-α. These demonstrate the importance of NRIP in AChR cluster formation during NMJ formation and maintenance as a novel AChR structural component.