NRG1 forward and backsignaling – differential recruitment of LIMK1?

NRG1-mediated canonical forward signaling proceeds via ErbB receptors. In addition to canonical forward signaling, backsignaling via the NRG1-ICD has been described in cultured cells (Bao et al., 2003; Mei and Xiong, 2008; Talmage, 2008; Chen et al., 2010; Fazzari et al., 2014; Mei and Nave, 2014). In addition to transcription factor functions after nuclear translocation (Bao et al., 2003), direct binding of the NRG-ICD to the serine/threonine kinase LIM kinase 1 (LIMK1) was observed (Wang et al., 1998). LIMK1 phosphorylates the actin depolymerizing factor cofilin and serves as a key regulator of actin cytoskeleton dynamics (Arber et al., 1998; Yang et al., 1998).

LIMK1 has been implicated in synaptic maturation (Huang et al., 2000) and shuttles between the cytoplasm and the nucleus (Yang and Mizuno, 1999).

To further exploit transgenic mouse models, including backsignaling-deficient HA-Nrg1^GIEF mice, for the investigation of forward and backsignaling-associated mechanisms, a comparative western blot analysis was performed using conditional Stop-Nrg1*NEX-Cre as well as “global” HA-Nrg1-tg and HA-Nrg1^GIEF mice. In agreement with previous findings, ErbB4 receptor phosphorylation correlated with the expression levels of different HA-NRG1 variants. As before, levels of total ErbB4 protein were reduced in all transgenic samples (Fig. 31A). Western blot analysis of hippocampal protein lysates used to examine effects in two major signaling pathways (Raf–MEK–ERK and PI3K–AKT–S6K pathways) downstream of NRG1/ErbB4

forward signaling in cultured cells (Mei and Xiong, 2008; Mei and Nave, 2014), showed that in mice with chronic overexpression of full length HA-NRG1 (HA-Nrg1-tg and Stop-Nrg1*NEX-Cre mice) MAPK or PI3K signaling was not activated (Fig. 31B).

However, both pathways were stimulated in HA-Nrg1^GIEF mice (Fig. 31B). This finding suggests more potent forward signaling functions of NRG1 NTF in HA-Nrg1^GIEF mice, even under chronic conditions. In line with this finding, the NRG1 NTF was localized to axons of projection neurons with a higher abundance than the full length CRD-NRG1 (Fig. 31B), similar to findings in the PNS (Velanac et al., 2011). Thus, the NRG1 NTF more likely reaches presynapses to signal to postsynaptic ErbB4 in interneurons.

Although the precise mechanisms are unknown, perturbed dendrite and axon growth of cortical neurons in CRD-NRG1 mouse mutants are thought to be mediated by NRG1-ICD backsignaling (Chen et al., 2008, 2010).

Immunostaining of paraffin sections from Stop-Nrg1*NEX-Cre mice with the anti-HA antibody revealed that HA-NRG1 accumulated in the somatodendritic compartment, whereas transport into the axonal and presynaptic compartment seemed to be limited (Fig. 31C). This lead to the working hypothesis that abnormal accumulation of full length HA-NRG1 in the somatodendritic compartment could lead to abnormal NRG1-ICD mediated backsignaling (Fig. 24C), with LIMK1 and cofilin as possible targets. Thus, hippocampal protein lysates from conditional Stop-Nrg1*NEX-Cre, as well as “global” HA-Nrg1-tg and HA-Nrg1^GIEF mice were tested for LIMK1 and cofilin phosphorylation.

To verify the identity of the LIMK1 band, protein lysates from LIMK1^-/- mice were included in the analysis. This analysis revealed that threonine residue 508 (important for the regulation of LIMK1 activity) was hypophosphorylated in Stop-Nrg1*NEX-Cre mice, indicating reduced LIMK1 activity (Fig. 31C). In contrast, the phosphorylation status of T508 was unaltered in HA-Nrg1-tg and HA-Nrg1^GIEF mice. Expression of LIMK1 protein was unchanged in all transgenic NRG1 mouse models. As expected this band was absent in protein lysates from LIMK1^-/- mice (Fig. 31C). Examination of the phosphorylation status of cofilin at serine 3 revealed increased Ser3 phosphorylation in all NRG1 transgenic models analyzed (Fig. 31C). Conditional Stop-Nrg1*NEX-Cremice showed stronger Ser3 phosphorylation than “global” HA-Nrg1-tg mice, which expressed the same HA-NRG1 variant even at higher levels.

Unexpectedly, backsignaling-deficient HA-Nrg1^GIEF mice showed the strongest Ser3 phosphorylation (Fig. 31C), implicating that NRG1 might regulate cofilin activity also via distinct forward directed signaling events. In none of the transgenic mouse models we did observe dysregulation of total cofilin protein (Fig. 31C).

Fig. 31: A comparative analysis of NRG1 transgenic mouse models reveals differences in forward and backsignaling. (A) (left) Western blot analysis of hippocampal protein lysates of WT, conditional Stop-Nrg1*NEX-Cre and global HA-Nrg1-tg and HA-Nrg1^GIEF mice reveals drastic differences in HA-CRD-NRG1 and HA-NRG1^GIEF expression levels that correlates with ErbB4 receptor hyperphosphorylation levels, in line with our working hypothesis (right). ErbB4 total protein is reduced in all NRG1 transgenic mouse models. (B) (left) Western blot analysis of hippocampal protein lysates (as in A) shows increased MAPK and AKT phosphorylation in HA-Nrg1^GIEF mice, but not in full length HA-CRD-NRG1 transgenic models, suggesting that the processed N-terminal fragment of CRD-NRG1 (containing the EGF-like domain) is the active part in canonical forward signaling. (Center) Illustration demonstration HA-NRG1^GIEF-mediated canonical forward signaling. (right) In line with this hypothesis, the HA-NRG1^GIEF fragment was found in axonal structures in HA-Nrg1^GIEF mice (Velanac et al., 2011). (C) (left) Western blot analysis of hippocampal protein lysates (as in A,B) reveals reduced LIMK1 phosphorylation in Stop-Nrg1*NEX-Cre mice, but not in ‘global’ models. LIMK1^-/- brain lysates were used to verify LIMK1 antibody reactivity.

Cofilin phosphorylation is increased in all HA-NRG1 transgenic models. Total LIMK1 and cofilin protein levels are unaltered in HA-NRG1 transgenic models. (Center) LIMK1 is thought to mediate CRD-NRG1 backsignaling via the NRG1-ICD, potentially leading to altered

dendritic spine dynamics by regulating the actin depolymerization activity of cofilin. However, backsignaling-deficient HA-Nrg1^GIEF mice also show increased cofilin phosphorylation. (right) Fluorescent immunostaining for HA-NRG1 on a coronal paraffin section (bregma -1.7) of a Stop-Nrg1*NEX-Cre mouse shows accumulation of HA-CRD-NRG1 in the somatodendritic compartment and apical dendrites (arrow heads). -actin was used as a loading control in all western blot experiments. FL, full length; kDa, kilodalton; IN, interneuron; Term, Nrg1 N-terminal fragment.

3.16 HA-NRG1 is enriched in synaptosomes of

mice