Loss of Navβ4-Mediated Regulation of Sodium Currents in Adult Purkinje Neurons Disrupts Firing and Impairs Motor Coordination and Balance

Abstract

SUMMARY The resurgent component of voltage-gated Na+ (Nav) currents, INaR, has been suggested to provide the depolarizing drive for high frequency firing and to be generated by voltage-dependent Nav channel block (at depolarized potentials) and unblock (at hyperpolarized potentials) by the accessory Navβ4 subunit. To test these hypotheses, we examined the effects of the targeted deletion of Scn4b (Navβ4) on INaR and on repetitive firing in cerebellar Purkinje neurons. We show here that Scn4b−/− animals have deficits in motor coordination and balance and that firing rates in Scn4b−/− Purkinje neurons are markedly attenuated. Acute, in vivo shRNA-mediated “knockdown” of Navβ4 in adult Purkinje neurons also reduced spontaneous and evoked firing rates. Dynamic clamp-mediated addition of INaR partially rescued firing in Scn4b−/− Purkinje neurons. Voltage-clamp experiments revealed that INaR was reduced (by ~50%), but not eliminated, in Scn4b−/− Purkinje neurons, revealing that additional mechanisms contribute to generation of INaR.

Effects of FGF14 and NaVβ4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons

“…Thus, Na V β4 may do little to modulate currents in WT Purkinje cell bodies. Notably, however, the present results differ from those reported by Ransdell et al (2017), in which Na V β4 was deleted using a different genetic approach and relative resurgent current was decreased but not eliminated. This discrepancy may be due to distinct compensatory mechanisms following the deletion of the protein using different approaches, or to different experimental conditions.…”

“…In all WT cells, regardless of the concentration of subsaturating TTX, transient currents at 0 mV exceeded 2 nA, and resurgent currents flowed upon repolarization to −30 mV after a 10-ms step to +30 mV. In all Na V β4 −/− cells, transient currents were large and resurgent currents were also evident, confirming that resurgent current can remain in Purkinje cells in the absence of functional Na V β4 (Ransdell et al, 2017). Owing to high Na current densities and fast kinetics, however, the voltage-clamp of transient currents was not always ideal.…”

“…Heterologous expression of Na V β4 with pore-forming subunits, however, cannot reconstitute resurgent current (Chen et al, 2008; Aman et al, 2009; Theile et al, 2011). Additionally, when Na V β4 expression is reduced or abolished in cerebellar granule cells, dorsal root ganglion (DRG) neurons, or Purkinje cells (Bant and Raman, 2010; Barbosa et al, 2015; Ransdell et al, 2017), resurgent current is decreased but not eliminated, raising the question of what other proteins directly or indirectly influence open-channel block.…”

Atxn2-CAG100-KnockIn mouse spinal cord shows progressive TDP43 pathology associated with cholesterol biosynthesis suppression

“…As novel insights into the progressive spinal cord pathology in this SCA2 model, Table 1 also highlights progressively reduced expression for several known neurodegeneration genes: firstly, Cdr1 (encoding Cerebellar Degeneration Related Protein 1, aka CDR62 or CDR34 or Yo-antigen) downregulation relates to its well-known autoimmune depletion as a cause of paraneoplastic ataxia [18] - it is interesting to note that Cdr1 expression is induced by the myelination factor Prion protein [172]; secondly, the Spinocerebellar Ataxia gene and voltage-gated potassium channel Kcna2 , which is preferentially expressed in afferent synapses onto the degenerating neurons [73, 229]; it is interesting to note that the parallel reduction of Kctd3 and Kctd9 affects two factors with potassium channel tetramerization domains; thirdly, the ALS disease gene Kif5a [20, 130] and its interactors Kif5b and Kif5c , which encode factors of axonal transport; Kif5a clusters with the progressive dysregulations of Uhmk1 (aka Ser/Thr-Protein kinase KIS) and Ina (aka internexin neuronal intermediate filament) in Table 1, since these factors relate to ribonucleoprotein and stress granule transport [24, 52, 108]. In the same context, the progressive expression downregulation of Hecw1 seems relevant, since this ubiquitination enzyme is responsible for the degradation of the ALS disease protein SOD1, is sequestrated into the cytosolic aggregates in ALS neurons, and its mutation leads to ALS-like phenotypes in mouse [123, 237]; fourth, the downregulation of Ano3 is important in view of its impact on tremor and dystonia [30, 196]; similarly, the reduced expression of Gnal encoding the G-protein G(olf) alpha, and of Rgs7bp encoding R7bp as general regulator of G-protein signaling appears relevant, in view of Gnal mutations triggering dystonia type 25 and the key role of R7bp in spinal afferents [51, 104, 141]; fifth, the insidious reduction of Scn4b mRNA seems relevant, given that Scn4b -null mice show motor coordination and balance deficits [158], that Scn4b expression depends on GABA-A signaling [150] and that Scn4b depletion was also observed in the striatum affected by polyglutamine-neurotoxicity due to Huntington’s disease mutation [139]. In view of the importance of some of these factors, validation of their dysregulation expression was done by RT-qPCR (Suppl.…”

Voltage-Gated Sodium Channel β Subunits and Their Related Diseases

“…Scn4b -null mice display deficits in balance and motor coordination and resurgent sodium current in null Purkinje neurons was reduced by approximately 50 percent. This was further validated using in vivo short hairpin RNA knockdown of β4 in adult Purkinje neurons (Ransdell et al 2017). Overexpression of the β4 subunit in Neuro2a cells results in increased neurite outgrowth, dendrite formation, and filopodia-like protrusions, suggesting a role for β4 in neuronal pathfinding and migration (Oyama et al 2006).…”