Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease.

Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFdelta/delta mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFdelta/delta mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. RESULTS: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFdelta/delta mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFdelta/delta mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFdelta/delta mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFdelta/delta mice, axon outgrowth is significantly reduced compared to wild-type littermates. CONCLUSIONS: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses.

Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation. Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques, in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions, electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and function.

The temperature-sensitive ion channel TRPV2 is endogenously expressed and functional in the primary sensory cell line F-11.

In sensory neurons heat is transduced by a subfamily of TRP channels sharing sequence homology with the capsaicin-sensitive vanilloid receptor subtype 1 (TRPV1), but differing in their thermal response thresholds. To identify a neuronal cell line endogenously expressing noxious heat-transducing ion channels, we examined F-11 cells, a hybridoma derived from rat dorsal root ganglia and mouse neuroblastoma. Using RT-PCR, transcripts homologous to TRPV2 and TRPV4, but not to TRPV1 or TRPV3, were found. We isolated a full-length cDNA of 2.4 kb coding for a 757-amino acid protein corresponding to mouse TRPV2, which was further characterized by immunocytochemistry and electrophysiology. Using the whole-cell patch-clamp technique, we observed a heat-evoked increase in outward and inward currents with a threshold of 51.6 +/- 0.2 degrees C. The current-voltage relationship stimulated by a temperature of 52 degrees C was characterized by an outward rectification with a reversal potential close to -10 mV. Heat-evoked currents could be inhibited by ruthenium red. There was no activation by stimulation with capsaicin or 2-aminoethoxydiphenyl borate. Our results indicate that F-11 cells express functional noxious heat-sensitive TRPV2 channels. Thus, we propose that F-11 cells represent a valuable in vitro model to characterize the properties of TRPV2 in a native neuronal environment.