A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Abstract

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin.

We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with β-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates.

BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.

Introduction

Spinal muscular atrophy (SMA) is an autosomal recessive disorder associated with the loss of alpha motor neurons in the brain stem and spinal cord [1]. SMA is classified into three types (I–III) based on the age of onset and clinical severity [2], and all are caused by genetic deletions or loss-of-function mutations in the survival of motor neurons gene, SMN1 [3], [4], [5], [6]. A second SMN gene, SMN2, produces predominantly an exon 7-deleted and functionally defective form of the protein due to a single point mutation in exon 7 [4], [7]. SMN is a ubiquitously expressed protein found in both the cytoplasm and nucleus. In the nucleus, SMN localises within discrete nuclear bodies termed “gems” (Gemini of Cajal (or coiled) bodies) [8], [9]. SMN forms a stable complex with gemins 2–7. This SMN complex is necessary for the correct assembly of small nuclear RNPs (snRNPs). In addition, the SMN complex associates with the Sm core proteins, unr-interacting protein (unrip), a novel 175 kDa protein (p175) and the heat shock cognate protein (hsc70) [10]. The latter complex was found in both the cytoplasm and the nucleus and mediates the ATP-dependent assembly of spliceosomal U snRNPs [10]. The presence of SMN in these ribonucleoprotein complexes suggests a role for SMN in mRNA biogenesis [11].

Another intriguing role for SMN is its involvement in axonal transport [12]. Early indications of the presence of SMN in neuronal dendrites [13], [14], [15] were supported by more recent evidence for SMN in growth-cone and filopodia-like structures in both neuronal and glial cells [16]. The active transport of SMN along axons to growth cones has been clearly demonstrated in a study that showed the existence of both β-actin mRNA and SMN in axonal processes [17]. SMN interacts with the mRNA-binding protein, hnRNP-R, which in turn binds to the 3′-UTR of β-actin mRNA [18]. This interaction is required for efficient transport of β-actin mRNA to growth cones of motor neurons [12]. It is not clear, however, whether SMN mediates this transport on its own or as a complex with gemins, similar to snRNP complexes. The precise mechanism by which SMN-assisted β-actin mRNP transport might enable correct axonal outgrowth and neuromuscular junction formation is far from clear.

Localisation of β-actin mRNA and protein to the growth cone is necessary for axonal outgrowth [17]. The dynamic actin cytoskeleton is the driving force for neuronal outgrowth and growth cone motility [19]. Regulation of actin dynamics is achieved by a set of actin-binding proteins. One of these is profilin, which was shown to bind to the proline-rich region of SMN [20]. Profilin can sequester actin monomers or promote polymerisation by desequestering of actin from the thymosin/β-actin pool and by adding these monomers to the free barbed ends of microfilaments [21]. Profilin also interacts with phosphatidylinositol 4,5-bisphosphate [22] and with proline-rich sequences in Ena/VASP proteins [23], [24], N-WASP [25] and diaphanous-related formins [26]. Five profilin isoforms have been identified in mammals, profilins I, IIa, IIb, III and IV, of which IIa and IIb arise from alternative splicing [27], [28], [54]. Profilin I is expressed ubiquitously, while profilin IIa is the major isoform in neuronal tissues both during development and in adult life [28]. Profilin is found throughout the cell body and is concentrated at active cell edges [29], [30]. Profilin is also found in gems and Cajal bodies in the nucleus where it co-localises with SMN, p-80 coilin and the Sm core proteins [20].

In the present study, we show the first evidence that SMN functions in a complex with gemins in the neurite cytoplasm. We have also explored what role the actin-binding protein, profilin, might have in mediating the effects of SMN on neuronal pathfinding and have studied profilin-binding by pathogenic SMN missense mutants to assess its role in SMA pathogenesis.