Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS

Abstract

Dysfunction of the RNA-binding protein, TDP-43, is strongly implicated as a causative event in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 is normally found in the nucleus and pathological hallmarks of ALS include the presence of cytoplasmic protein aggregates containing TDP-43 and an associated loss of TDP-43 from the nucleus. Loss of nuclear TDP-43 likely contributes to neurodegeneration. Using Drosophila melanogaster to model TDP-43 loss of function, we show that reduced levels of the voltage-gated calcium channel, cacophony, mediate some of the physiological effects of TDP-43 loss. Null mutations in the Drosophila orthologue of TDP-43, named TBPH, resulted in defective larval locomotion and reduced levels of cacophony protein in whole animals and at the neuromuscular junction. Restoring the levels of cacophony in all neurons or selectively in motor neurons rescued these locomotion defects. Using TBPH immunoprecipitation, we showed that TBPH associates with cacophony transcript, indicating that it is likely to be a direct target for TBPH. Loss of TBPH leads to reduced levels of cacophony transcript, possibly due to increased degradation. In addition, TBPH also appears to regulate the inclusion of some alternatively spliced exons of cacophony. If similar effects of cacophony or related calcium channels are found in human ALS patients, these could be targets for the development of pharmacological therapies for ALS.

Exploring the Function of Exon 7 in Drosophila Cacophony in Relation to ALS

“…In the Morton Lab fly model for TDP-43 loss of function (TBPH null), loss of TBPH results in reduced levels of cacophony (Chang et al, 2014). The loss of function model showed late pupal lethality, and defective larval crawling that can be rescued by expressing cacophony selectively in motor neurons, as well as rescuing with TBPH (Chang et al, 2014).…”

“…Reduced levels of cacophony and altered splicing due to the loss of TBPH, and the presence of this protein at the active zone of the neuromuscular junction suggest that cacophony is a target for TDP-43, and is potentially connected to motor neuron degradation in patients with ALS (Kawasaki, Zou, Xu, & Ordway, 2004). Altered RNA splicing seen in TBPH null flies directly resulted in a lower level of transcripts that contain exon 7 (Chang et al, 2014). Exon 7 is believed to code for a section of the cytoplasmic C-terminal tail of cacophony, a region that is "known to affect inactivation and calcium regulation" (Macleod et al, 2006).…”

“…Exon 7 is believed to code for a section of the cytoplasmic C-terminal tail of cacophony, a region that is "known to affect inactivation and calcium regulation" (Macleod et al, 2006). "Of the 15 predicted cacophony transcripts on FlyBase only one, cac-RM, lacks exon 7 suggesting it plays a significant physiological role," though the specific function is unknown (Chang et al, 2014). Absence of TBPH results in larvae that have reduced locomotion and are late pupal lethal.…”

“…In the Morton Lab fly model for TDP-43 loss of function (TBPH null), loss of TBPH results in reduced levels of cacophony (Chang et al, 2014). The loss of function model showed late pupal lethality, and defective larval crawling that can be rescued by expressing cacophony selectively in motor neurons, as well as rescuing with TBPH (Chang et al, 2014). Reduced levels of cacophony and altered splicing due to the loss of TBPH, and the presence of this protein at the active zone of the neuromuscular junction suggest that cacophony is a target for TDP-43, and is potentially connected to motor neuron degradation in patients with ALS (Kawasaki, Zou, Xu, & Ordway, 2004).…”

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“…MNs expressing EKI were identified by co-expression of a red fluorescent 85 protein (RFP) tag attached to the promoter and a green fluorescent protein (GFP) tag 86 attached to the EKI construct [22]. 87 Figure 1. Schematic of the larval preparation.…”