Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits

Abstract

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of β-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Fig. 1.

SMN interacts with HuD. (A) List of proteins that coimmunoprecipitate with SMN from cortical neurons with an FDR ∼1%. All peptides with a Mascot score >35 are listed. All proteins listed were found in three biological replicates. Unique peptides observed for each protein are listed in Table S1. (B) Mass spectrum of a peptide derived from HuD identified in SMN IP from cortical neurons. HuD protein is a strong interactor, with a protein score of 75 and an FDR of ≤1%. (C) IP from spinal cord lysates using the SMN, HuD, or IgG antibody. A 38-kDa band representing SMN protein is observed in the HuD IPs, and a 40- to 45-kDa band specific for HuD is seen in the SMN IPs. (D) Immunofluorescence with anti-SMN (green) and anti-HuD (red) showing colocalization of the HuD protein with SMN granules along the motor neuron axon (0.1-μm section). (E) Detail of D showing colocalization of SMN with HuD. Arrows indicate fully overlapping signals. (F) Graph of the mean ± SEM percentage of SMN granules that colocalize with the HuD signal or synaptophysin signal (P < 0.005, Student t test; n = 7–10 per group).

Fig. 2.

Cpg15 mRNA interacts with HuD and SMN in vitro and in vivo. RNA was isolated from the HuD-IP (A) and SMN-IP (B) complexes of cortical neurons. RT-PCR was performed using primers specific for HuD, SMN, cpg15, β-actin, Homer, GAP43, Tau, EphA4, and GAPDH (n = 6, from three independent experiments and two technical replicates; Table S2). Data represent values normalized to IgG-IPs. (C) cpg15 mRNA (red) colocalizes with SMN protein (green) in motor neuron axons. (Inset) Arrows show colocalization of SMN and cpg15 mRNA within the axon.

Fig. 3.

Cpg15 mRNA distribution and local translation. (A) cpg15 mRNA expression decreases in the neurites when SMN levels are reduced. Cortical neurons were infected with lentivirus carrying either SMN shRNA (shSMN) or the control empty vector (CTL). RNA was isolated from both compartments of the Boyden chamber after 10DIV. Equal amounts of RNA was used to prepare cDNAs and quantitative real-time RT-PCR was performed using primers specific for SMN, cpg15, β-actin, and GAPDH mRNA. Results are normalized to control conditions for the cell body and the neurites separately. *P < 0.0001, Student t test; n = 12, from six independent biological sets. Data are presented as mean ± SEM. (B) cpg15 is locally translated in the growth cones. Representative images demonstrate the fluorescence recovery after photobleaching (FRAP) in distal hippocampal axons transfected with dGFP^myr-cpg15 over a period of 12 min in the absence (Upper) or presence (Lower) of 100 μM CHX. (Scale bar: 10 μm.) (C) Quantification of GFP intensity in the distal growth cones during prebleaching and postbleaching of hippocampal neurons in the absence (CTL, black line) or presence (+CHX, red line) of CHX. For each time point, the data represent an average percentage of prebleach intensity ± SEM, with the prebleach intensity normalized to 100 (n = 3 for the +CHX group; n = 5 for the CTL group). Significant recovery was observed by two-way ANOVA when comparing the CTL and +CHX groups at each time point: *P < 0.05 from 6.7 to 7.3 min; **P < 0.01 from 7.7 to 12 min.

Fig. 4.

Cpg15 partially rescues SMN deficiency. (A–C) Lateral-view representative images of Tg(hb9:GFP) embryos at 28 hpf uninjected (A), injected with 9 ng of smn MO (B), and injected with 9 ng of smn MO and 200 ng of full-length human cpg15 mRNA (C). (D) Full-length human cpg15 rescues motor axon defects caused by a reduction of Smn in zebrafish. Tg(hb9:GFP) zebrafish injected with 9 ng of smn MO and scored at 28 hpf using previously published criteria (29) resulted in a distribution of fish with motor nerve defects (n = 260 fish, 5,200 nerves, three injections). Coinjection of 9 ng of smn MO with 200 pg of full-length human cpg15 mRNA was able to partially rescue the nerve defects (n = 326 fish, 6,520 nerves; P < 0.01). The distribution of larval classifications (severe, moderate, mild, and no defects) was analyzed using the Mann–Whitney nonparametric rank test.