HuD and the Survival Motor Neuron Protein Interact in Motoneurons and Are Essential for Motoneuron Development, Function, and mRNA Regulation

Abstract

Motoneurons establish a critical link between the CNS and muscles. If motoneurons do not develop correctly, they cannot form the required connections, resulting in movement defects or paralysis. Compromised development can also lead to degeneration because the motoneuron is not set up to function properly. Little is known, however, regarding the mechanisms that control vertebrate motoneuron development, particularly the later stages of axon branch and dendrite formation. The motoneuron disease spinal muscular atrophy (SMA) is caused by low levels of the survival motor neuron (SMN) protein leading to defects in vertebrate motoneuron development and synapse formation. Here we show using zebrafish as a model system that SMN interacts with the RNA binding protein (RBP) HuD in motoneurons in vivo during formation of axonal branches and dendrites. To determine the function of HuD in motoneurons, we generated zebrafish HuD mutants and found that they exhibited decreased motor axon branches, dramatically fewer dendrites, and movement defects. These same phenotypes are present in animals expressing low levels of SMN, indicating that both proteins function in motoneuron development. HuD binds and transports mRNAs and one of its target mRNAs, Gap43, is involved in axonal outgrowth. We found that Gap43 was decreased in both HuD and SMN mutants. Importantly, transgenic expression of HuD in motoneurons of SMN mutants rescued the motoneuron defects, the movement defects, and Gap43 mRNA levels. These data support that the interaction between SMN and HuD is critical for motoneuron development and point to a role for RBPs in SMA.Key words: HuD; motoneuron; RNA binding protein; spinal muscular atrophy; survival motor neuron; zebrafish IntroductionNeuronal development is controlled at many levels. This includes transcriptional programs that dictate the type of neuron, RNA transport for localized RNA translation, and downstream pathways important for the formation of neuronal structures necessary for their function, such as axons, dendrites, and synapses. The disruption of any of these steps results in a motoneuron that either does not develop at all or one that develops, but does not function properly. For motoneurons, the initial developmental This work was supported by National Institutes of Health R56NS050414 and RO1NS098780 and CureSMA to C.E.B., and the Greater Milwaukee Foundation Shaw Scientist Award 133-AAA2656 to M.W. We thank Dr. Wilfried Rossoll for the mCherry-HuD construct; and the fish facility staff for fish care.The authors declare no competing financial interests. Significance StatementIn zebrafish models of the motoneuron disease spinal muscular atrophy (SMA), motor axons fail to form the normal extent of axonal branches and dendrites leading to decreased motor function. SMA is caused by low levels of the survival motor neuron (SMN) protein. We show in motoneurons in vivo that SMN interacts with the RNA binding protein, HuD. Novel mutants reveal that HuD is also necessary for motor axonal branch and dendrite form...

The role of survival motor neuron protein (SMN) in protein homeostasis

“…SMN acts as a molecular chaperone for the binding of RBPs to mRNA transcripts as well the RBPs’ binding to the cytoskeleton and subsequent localisation, as evidenced by disruption of these processes in SMN deficiency [ 121 ]. Both IMP1 and HuD have been shown to influence the localisation and translation of β-actin and GAP-43 mRNA transcripts, which are in turn both necessary for correct axonal growth [ 113 , 118 , 122 ]. Indeed, SMN knockdown leads to a reduction of HuD in the axonal compartment [ 113 ], while knockdown of HuD in zebrafish leads to a similar phenotype to SMN knockdown of shorter axons [ 122 ].…”

“…Both IMP1 and HuD have been shown to influence the localisation and translation of β-actin and GAP-43 mRNA transcripts, which are in turn both necessary for correct axonal growth [ 113 , 118 , 122 ]. Indeed, SMN knockdown leads to a reduction of HuD in the axonal compartment [ 113 ], while knockdown of HuD in zebrafish leads to a similar phenotype to SMN knockdown of shorter axons [ 122 ]. Further experiments using FLAG-tagged SMN in NSC-34 cells [ 123 ] identified SMN as a binding partner for several species of non-coding RNAs, including snRNAs, snoRNAs and ribosomal RNAs, which were expected due to SMN’s known role in RNA processing, as well as miRNAs and tRNAs.…”

Overlapping activities of ELAV/Hu RNA binding proteins specify multiple neural alternative splicing programs

“…In mice, knockout of HuD causes behavioral defects [83] while HuB/D double mutants exhibit seizures [19]. Zebrafish knockouts of HuD exhibit neural differentiation and behavioral defects [84], and a C. elegans Elav homolog is required for synaptic physiology [85]. Overall, ELAV/Hu proteins are implicated in a variety of neural gene regulation and behavioral regimes [86,87].…”

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals

“…SMN has been shown to function in the assembly of Sm proteins onto snRNAs as well as the assembly of Sm/Lsm proteins onto the U7 small nuclear ribonucleic acid (snRNA) (30)(31)(32)(33)(34). SMN has also been implicated in the assembly of RNP complexes that play a role in transport and translational control of mRNA in axons (30,35,36). SMN mutations equivalent to those found in SMA patients have been examined in species other than mammals.…”