No abstract

Though there is compelling evidence that de-innervation of neuromuscular junctions (NMJ) occurs early in amyotrophic lateral sclerosis (ALS), defects arising at synapses in the spinal cord remain incompletely understood. To investigate spinal cord synaptic dysfunction, we took advantage of a zebrafish larval model and expressed either wild type human TARDBP (wt TARDBP ) or the ALS-causing G348C variant (mut TARDBP ). The larval zebrafish is ideally suited to examine synaptic connectivity between descending populations of neurons and spinal cord motoneurons as a fully intact spinal cord is preserved during experimentation. Here we provide evidence that the tail-beat motor pattern is reduced in both frequency and duration in larvae expressing mut TARDBP . In addition, we report that motor-related synaptic depolarizations in primary motoneurons of the spinal cord are shorter in duration and fewer action potentials are evoked in larvae expressing mut TARDBP . To more thoroughly examine spinal cord synaptic dysfunction in our ALS model, we isolated AMPA/kainate-mediated glutamatergic miniature excitatory post-synaptic currents in primary motoneurons and found that in addition to displaying a larger amplitude, the frequency of quantal events was higher in larvae expressing mut TARDBP when compared to larvae expressing wt TARDBP . In a final series of experiments, we optogenetically drove neuronal activity in the hindbrain and spinal cord population of descending ipsilateral glutamatergic interneurons (expressing Chx10 ) using the Gal4-UAS system and found that larvae expressing mut TARDBP displayed abnormal tail-beat patterns in response to optogenetic stimuli and augmented synaptic connectivity with motoneurons. These findings indicate that expression of mut TARDBP results in functionally altered glutamatergic synapses in the spinal cord.

Mutations in CHCHD10 and CHCHD2, encoding two paralogous mitochondrial proteins, have been identified in cases of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Parkinson's disease. Their role in disease is unclear, though both have been linked to mitochondrial respiration and mitochondrial stress responses. Here, we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish. We demonstrate that loss of either or both proteins leads to motor impairment, reduced survival and compromised neuromuscular junction integrity in larval zebrafish. Compensation by Chchd10 was observed in the chchd2−/− model, but not by Chchd2 in the chchd10−/− model. The assembly of mitochondrial respiratory chain Complex I was impaired in chchd10−/− and chchd2−/− zebrafish larvae, but unexpectedly not in a double chchd10−/− and chchd2−/− model, suggesting that reduced mitochondrial Complex I cannot be solely responsible for the observed phenotypes, which are generally more severe in the double KO. We observed transcriptional activation markers of the mitochondrial integrated stress response (mt‐ISR) in the double chchd10−/− and chchd2−/− KO model, suggesting that this pathway is involved in the restoration of Complex I assembly in our double KO model. The data presented here demonstrates that the Complex I assembly defect in our single KO models arises independently of the mt‐ISR. Furthermore, this study provides evidence that both proteins are required for normal vertebrate development.