Role of Nav1.9 in activity-dependent axon growth in motoneurons

Abstract

Spontaneous neural activity promotes axon growth in many types of developing neurons, including motoneurons. In motoneurons from a mouse model of spinal muscular atrophy (SMA), defects in axonal growth and presynaptic function correlate with a reduced frequency of spontaneous Ca(2+) transients in axons which are mediated by N-type Ca(2+) channels. To characterize the mechanisms that initiate spontaneous Ca(2+) transients, we investigated the role of voltage-gated sodium channels (VGSCs). We found that low concentrations of the VGSC inhibitors tetrodotoxin (TTX) and saxitoxin (STX) reduce the rate of axon growth in cultured embryonic mouse motoneurons without affecting their survival. STX was 5- to 10-fold more potent than TTX and Ca(2+) imaging confirmed that low concentrations of STX strongly reduce the frequency of spontaneous Ca(2+) transients in somatic and axonal regions. These findings suggest that the Na(V)1.9, a VGSC that opens at low thresholds, could act upstream of spontaneous Ca(2+) transients. qPCR from cultured and laser-microdissected spinal cord motoneurons revealed abundant expression of Na(V)1.9. Na(V)1.9 protein is preferentially localized in axons and growth cones. Suppression of Na(V)1.9 expression reduced axon elongation. Motoneurons from Na(V)1.9(-/-) mice showed the reduced axon growth in combination with reduced spontaneous Ca(2+) transients in the soma and axon terminals. Thus, Na(V)1.9 function appears to be essential for activity-dependent axon growth, acting upstream of spontaneous Ca(2+) elevation through voltage-gated calcium channels (VGCCs). Na(V)1.9 activation could therefore serve as a target for modulating axonal regeneration in motoneuron diseases such as SMA in which presynaptic activity of VGCCs is reduced.

Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice

“…Isolation of total RNA, reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR) were performed as previously described with few changes (57). Total RNA from primary cultured mouse kidney cells and mouse kidney tissue was isolated and purified with the RNeasy Mini Kit (QIAGEN).…”

“…Oligo 6.0 software (MedProbe) served as program to select intron-spanning primers targeting the mus musculus cathepsin D g ene (NM_009983.2; 330-forward 5′-GCCGCAGTGTTTCACAG-3′; 479-reverse 5′-TGAGCCGTAGTGGATGTCAA-3′; amplification product: 169 bp). Optimized primers for the two housekeeping reference genes, GAPDH and β-actin, have been used as published earlier (57): GAPDH: NM_008084; 205-forward 5′-GCAAATTCAACGGCACA-3′; 337-reverse 5′-CACCAGTAGACTCCACGAC-3′; amplification product: 141 bp; β-actin: NM_007393; forward 5′-GCCAACCGTGAAAAGATGAC-3′; reverse 5′-GGCGTGAGGGAGAGCATAG-3′. In order to verify the specificity of the PCR products gel electrophoresis, melting curve analysis and ‘-RT’ (reverse transcriptase) as well as water control PCR reactions were conducted.…”

A de novo gain-of-function mutation in SCN11A causes loss of pain perception

“…Muscular weakness in the affected individuals might be attributed to a developmental role of the channel in motor neurons, as Na v 1.9 was shown to contribute to the spontaneous neural activity that promotes axon growth in these neurons 33,34 . Prominent hyperhidrosis and gastrointestinal malfunction in the affected individuals may be a consequence of autonomic dysfunction.…”

Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation

“…For knockdown of Smn, an Smn (5 ′ -GAAGAATGCCACAACTCCC -3 ′ ) shRNA sequence (Zhang et al 2008) was cloned into the pSIH-H1 shRNA vector (System Bioscience) and HEK293T cells were used to generate viruses as described previously (Lois et al 2002; Smn deficiency alters the axonal transcriptome www.rnajournal.org 1799 Subramanian et al 2012). Lentiviral knockdown experiments were performed by incubation of motoneurons before plating with either knockdown or control viruses containing empty pSIH-H1 for 10 min at room temperature.…”