Despite recent progress in the understanding of the role of SMN in snRNP assembly, there is no evidence so far that disturbance of this function is responsible for the development of motoneuron disease in SMA. We define a role of Smn and its binding partner hnRNP R in axonal growth in motoneurons. Furthermore, we show that dendrite growth is not disturbed in motoneurons from Smn mutant mice. We also found that Smn deficiency had no effect on motoneuron survival in cell culture. These data suggest that disturbances in axon growth and maintenance are the primary cellular defects that ultimately may lead to loss of motoneurons in SMA. Recently, it has been shown that reduction of Smn levels in zebrafish embryos causes axon-specific pathfinding defects in motoneuron development (McWhorter et al., 2003
). These data support our finding of specific axonal defects in Smn-deficient murine motoneurons.
In differentiating PC12 cells, overexpression of Smn as well as hnRNP R leads to enhanced neurite outgrowth. This effect depends on the interaction of these two proteins. They are colocalized in axons of cultured motoneurons and also in axons of motor nerves in adult mice (Rossoll et al., 2002
). SMN protein has been shown to be present in a stable complex with Gemin2–7 (for review see Meister et al., 2002
; Paushkin et al., 2002
) in the nucleus within specific structures called gemini of Cajal (coiled) bodies (“gems”). Interestingly, hnRNP R has not been identified as part of this complex. Gems seem to play an important role for snRNP assembly and RNA processing (Carvalho et al., 1999
; Young et al., 2000
; Sleeman et al., 2001
). In motoneurons, Smn is also localized in axons (Pagliardini et al., 2000
; Jablonka et al., 2001
; Fan and Simard, 2002
), but it does not colocalize with Gemin2 (Jablonka et al., 2001
), and instead colocalizes with hnRNP R in this part of the cell (Rossoll et al., 2002
). Others have observed Smn in association with cytoskeletal elements in spinal dendrites and axons (Bechade et al., 1999
; Pagliardini et al., 2000
). Recently, cytoskeletal-based active transport of SMN containing granules in neuronal processes and growth cones has been demonstrated in transfected cultured neurons (Zhang et al., 2003
). Binding of Smn to hnRNP R and localization of these proteins in motor axons (Rossoll et al., 2002
) suggest that Smn could be involved in the transport of specific mRNAs in motor axons.
Increasing evidence points to the importance of RNA localization and transport within polarized cells. Sorting of defined mRNA species to distinct subcellular regions is observed in many cell types, in particular in neurons (for review see Mohr and Richter, 2001
). The actin cytoskeleton plays an important role in axon initiation, growth, guidance, branching, and retraction, and also in synapse formation and stability (for review see Luo, 2002
). β-Actin protein is highly enriched in distal parts of axons and growth cones. Specific transport of β-actin mRNA to axons contributes to this distribution (Bassell et al., 1998
; Zhang et al., 1999
). Our observation that axon growth is significantly impaired in Smn−/−; SMN2
motoneurons (and that β-actin protein distribution and growth cone morphology is disturbed in these cells) points to an essential role of Smn and hnRNP R in this function. To investigate a possible direct effect on the transport of β-actin mRNA, we performed in situ hybridization experiments to localize actin transcripts in neurites of developing PC12 cells and primary motoneurons (). Our results suggest that wild-type Smn levels are required for accumulation of actin mRNA in growth cones of motoneurons. Furthermore, we show that hnRNP R mutants that cannot bind RNA or Smn exert a dominant-negative effect on mRNA translocation. This corresponds to reduced neurite growth in PC12 cell lines that express hnRNP R ΔRRM ( A). Our data show that the observed reduced distal β-actin protein localization is caused, at least to some extent, by a direct effect of Smn and hnRNP R on distal actin mRNA accumulation.
It has been shown that the localization of β-actin mRNA involves a 54-nt sequence within the 3′ UTR (termed zipcode) that is both sufficient and necessary for peripheral localization (Kislauskis et al., 1994
). Our findings indicate that hnRNP R can associate with full-length β-actin mRNA as well as the zipcode region, and that this interaction does not depend on a poly(A)+
tail. Because neither the lysozyme nor the IκBα control mRNAs are bound, specificity for this interaction can be anticipated. Two proteins, ZBP1 and ZBP2, which interact with the same zipcode domain of β-actin mRNA and seem to be involved in axonal transport of β-actin mRNA in chick forebrain (Zhang et al., 2001
; Gu et al., 2002
) or rat cortical neurons (Bassell et al., 1998
), have been identified. However, it is not clear so far whether these proteins need the hnRNP R–Smn complex for this effect, or whether motoneurons specifically require this complex either in concert or independently from ZBP1 and ZBP2. Many attempts have been made to identify Smn-interacting proteins (for review see Meister et al., 2002
; Paushkin et al., 2002
), but interaction with ZBP1 and ZBP2 has not been found by such efforts. Further experiments with mouse gene knockout models will need to show the specific requirement of ZBP1, ZBP2, and/or the hnRNP R/Q–Smn complex for axonal β-actin mRNA transport in motoneurons and other types of neurons.
It is possible that the role of hnRNP R–Smn in motoneurons goes beyond transport of β-actin mRNA. hnRNP Q has been implicated in regulation of mRNA stability (Grosset et al., 2000
), editing (Blanc et al., 2001
; Lau et al., 2001
), and splicing (Mourelatos et al., 2001
). Therefore, the precise function of these RNA-binding proteins in axons remains to be determined. For example, it could be that the interaction of β-actin mRNA and hnRNP R–Smn also controls stability of β-actin mRNA in motor growth cones. It could also be that this complex is part of a regulatory machinery that controls local actin protein translation in response to extracellular stimuli, which control growth cone migration, presynaptic differentiation, and functions at the motor endplate. This scenario appears as a tempting model to interpret the pathophysiology of SMA. Recently, it has been proposed that the primary function of actin in the growth cone is not propulsion, but is to act as a scaffold for regulatory molecules in the presynapse (Sankaranarayanan et al., 2003
). Specific defects in neurofilament distribution in motor axons, as well as defects of axonal sprouting and axonal growth, were observed in another mouse model (Cifuentes-Diaz et al., 2002
). Although we did not find a direct effect on the distribution of neurofilaments in axons, a relative accumulation in growth cones was observed. However, this appears as a consequence to reduced β-actin content in growth cones and reduced size of the axon terminals. In this scenario, proteins that normally are not present in presynaptic structures, such as neurofilaments and microtubules, appear more distal in growth cones. The reduced levels of actin suggest that functions such as growth cone movement and the release of synaptic vesicles (Doussau and Augustine, 2000
; Bloom et al., 2003
), which also require actin, might be disturbed in SMA, and thus contribute to the specific pathology of this disease. Defects in dynamic processes that are necessary for further maturation and function of the presynaptic part of the motor endplate could thus constitute a major part of the pathophysiology of SMA. Future experiments will have to show whether the hnRNP R–Smn complex also modulates synaptic excitability, and thus plays a role that goes beyond the observed effect on axon growth in motoneurons.