Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma

Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan–Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increase BMPR1B expression. In agreement, nerve growth factor treatment of cultured sympathetic neurons decreased Bmpr1a expression and increased Bmpr1b expression. Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB.

T-type Ca 2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca 2+ influx, and highly selective T-type Ca 2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca 2+ channels, prevented enhanced axon growth. T-type Ca 2+ channel-specific inhibitors eliminated Ca 2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca 2+ channels along sympathetic axons, which is required for enhanced axon growth.

Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.

Pseudorabies Virus Us9 Directs Axonal Sorting of Viral Capsids

ABSTRACT Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

Modulation of a Delayed-Rectifier K+ Current by Angiotensin II in Rat Sympathetic Neurons

It is well known that angiotensin II (Angio II) mimics most of the muscarinic-mediated excitatory actions of acetylcholine on superior cervical ganglion neurons. For instance, in addition to depolarization and stimulation of norepinephrine release, muscarinic agonists and Angio II modulate the M-type K+ current and the N-type Ca2+ current. We recently found that muscarinic receptors modulate the delayed rectifier current IKV as well. Therefore a whole cell patch-clamp experiment was carried out in rat cultured sympathetic neurons to assess whether Angio II modulates IKV. We found that Angio II increased IKV by about 30% with a time constant of approximately 30 s. In comparison, inhibition of M-current was faster (τ ∼ 8 s) and stronger (∼61%). Modulation of IKV was disrupted by the AT1 receptor-antagonist losartan but not by the AT2-antagonist PD123319. IKV enhancement was reduced by the G-protein inhibitor GDP-β-S, whereas current modulation remained unaltered after cell treatment with pertussis toxin. The peptidergic modulation of IKV was severely disrupted when internal ATP was replaced by its nonhydrolyzable analogue AMP-PNP. Angio II enhanced IKV and further reduced the stimulatory action of a muscarinic agonist on IKV. Likewise, the muscarinc agonist enhanced IKV and occluded the effect of Angio II on IKV. We have also found that the protein kinase C activator PMA enhanced IKV, thereby mimicking and further attenuating the action of Angio II on IKV. These results suggest that AT1 receptors by coupling to pertussis toxin–insensitive G proteins, stimulate an ATP-dependent and PKC-mediated pathway to modulate IKV.