Deletion of a specific exon in the voltage-gated calcium channel gene cacophony disrupts locomotion in Drosophila larvae.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein that regulates transcription, translation and alternative splicing of mRNA. We have shown previously that null mutations of the Drosophila ortholog, Tar DNA-binding homolog (tbph), causes severe locomotion defects in larvae that are mediated by a reduction in the expression of a type II voltage-gated calcium channel, cacophony (cac). We also showed that TDP-43 regulates the inclusion of alternatively spliced exons of cacophony; tbph mutants showed significantly increased expression of cacophony isoforms lacking exon 7, a particularly notable finding as only one out of the 15 predicted isoforms lacks exon 7. To investigate the function of exon 7, we generated Drosophila mutant lines with a deletion that eliminates exon 7. This deletion phenocopies many defects in tbph mutants: a reduction in cacophony protein (Dmca1A) expression, locomotion defects in male and female third instar larvae, disrupted larval motor output, and also reduced activity levels in adult male flies. All these defects were rescued by expression of cacophony transcripts containing exon 7. By contrast, expression of a cacophony cDNA lacking exon 7 resulted in reduced cacophony protein levels and failed to rescue larval locomotion.

Drosophila Exo70 Is Essential for Neurite Extension and Survival under Thermal Stress.

The octomeric exocyst complex governs the final step of exocytosis in both plants and animals. Its roles, however, extend beyond exocytosis and include organelle biogenesis, ciliogenesis, cell migration, and cell growth. Exo70 is a conserved component of the exocyst whose function in Drosophila is unclear. In this study, we characterized two mutant alleles of Drosophila exo70. exo70 mutants exhibit reduced synaptic growth, locomotor activity, glutamate receptor density, and mEPSP amplitude. We found that presynaptic Exo70 is necessary for normal synaptic growth at the neuromuscular junction (NMJ). At the neuromuscular junction, exo70 genetically interacts with the small GTPase ralA to regulate synaptic growth. Loss of Exo70 leads to the blockage of JNK signaling-, activity-, and temperature-induced synaptic outgrowths. We showed that this phenotype is associated with an impairment of integral membrane protein transport to the cell surface at synaptic terminals. In octopaminergic motor neurons, Exo70 is detected in synaptic varicosities, as well as the regions of membrane extensions in response to activity stimulation. Strikingly, mild thermal stress causes severe neurite outgrowth defects and pharate adult lethality in exo70 mutants. exo70 mutants also display defective locomotor activity in response to starvation stress. These results demonstrated that Exo70 is an important regulator of induced synaptic growth and is crucial for an organism's adaptation to environmental changes.SIGNIFICANCE STATEMENT The exocyst complex is a conserved protein complex directing secretory vesicles to the site of membrane fusion during exocytosis, which is essential for transporting proteins and membranes to the cell surface. Exo70 is a subunit of the exocyst complex whose roles in neurons remain elusive, and its function in Drosophila is unclear. In Drosophila, Exo70 is expressed in both glutamatergic and octopaminergic neurons, and presynaptic Exo70 regulates synaptic outgrowth. Moreover, exo70 mutants have impaired integral membrane transport to the cell surface at synaptic terminals and block several kinds of induced synaptic growth. Remarkably, elevated temperature causes severe arborization defects and lethality in exo70 mutants, thus underpinning the importance of Exo70 functions in development and adaptation to the environment.

Exploring the Interaction of Drosophila TDP-43 and the Type II Voltage-Gated Calcium Channel, Cacophony, in Regulating Motor Function and Behavior.

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neurodegenerative disease. The cause of the disease remains obscure, and as such there is no effective treatment or cure. Amyotrophic lateral sclerosis and other neurodegenerative diseases are frequently characterized by dysfunction of the RNA-binding protein, TDP-43. Using model systems to understand the mechanisms underlying TDP-43 dysfunction should accelerate identification of therapeutic targets. A recent report has shown that motor defects caused by the deletion of the Drosophila TDP-43 ortholog, tbph, are not driven by changes in the physiology at the neuromuscular junction. Rather, defective motor burst rhythmicity and coordination, displayed by tbph mutants, are rescued by genetically restoring a voltage-gated calcium channel to either motor neurons or just a single pair of neurons in the brain. If these effects are mirrored in human TDP-43 proteinopathies, these observations could open new avenues to investigate alternative therapeutic targets for these neurodegenerative diseases.

Restoration of Motor Defects Caused by Loss of Drosophila TDP-43 by Expression of the Voltage-Gated Calcium Channel, Cacophony, in Central Neurons.

Defects in the RNA-binding protein, TDP-43, are known to cause a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. A variety of experimental systems have shown that neurons are sensitive to TDP-43 expression levels, yet the specific functional defects resulting from TDP-43 dysregulation have not been well described. Using the Drosophila TDP-43 ortholog TBPH, we previously showed that TBPH-null animals display locomotion defects as third instar larvae. Furthermore, loss of TBPH caused a reduction in cacophony, a Type II voltage-gated calcium channel, expression and that genetically restoring cacophony in motor neurons in TBPH mutant animals was sufficient to rescue the locomotion defects. In the present study, we examined the relative contributions of neuromuscular junction physiology and the motor program to the locomotion defects and identified subsets of neurons that require cacophony expression to rescue the defects. At the neuromuscular junction, we showed mEPP amplitudes and frequency require TBPH. Cacophony expression in motor neurons rescued mEPP frequency but not mEPP amplitude. We also showed that TBPH mutants displayed reduced motor neuron bursting and coordination during crawling and restoring cacophony selectively in two pairs of cells located in the brain, the AVM001b/2b neurons, also rescued the locomotion and motor defects, but not the defects in neuromuscular junction physiology. These results suggest that the behavioral defects associated with loss of TBPH throughout the nervous system can be associated with defects in a small number of genes in a limited number of central neurons, rather than peripheral defects.SIGNIFICANCE STATEMENT TDP-43 dysfunction is a common feature in neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar dementia, and Alzheimer's disease. Loss- and gain-of-function models have shown that neurons are sensitive to TDP-43 expression levels, but the specific defects caused by TDP-43 loss of function have not been described in detail. A Drosophila loss-of-function model displays pronounced locomotion defects that can be reversed by restoring the expression levels of a voltage-gated calcium channel, cacophony. We show these defects can be rescued by expression of cacophony in motor neurons and by expression in two pairs of neurons in the brain. These data suggest that loss of TDP-43 can disrupt the central circuitry of the CNS, opening up identification of alternative therapeutic targets for TDP-43 proteinopathies.