Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.

Objective: To further characterize mitochondrial dysfunction in LRRK2 G2019S mutant Parkinson disease (PD) patient tissue (M-LRRK2 G2019S), determine whether ursodeoxycholic acid (UDCA) also exerts a beneficial effect on mitochondrial dysfunction in nonmanifesting LRRK2 G2019S mutation carriers (NM-LRRK2 G2019S), and assess UDCA for its beneficial effect on neuronal dysfunction in vivo.Methods: Intracellular adenosine 59-triphosphate (ATP) levels, oxygen consumption, and activity of the individual complexes of the mitochondrial respiratory chain as well as mitochondrial morphology were measured in M-LRRK2G2019S , NM-LRRK2 G2019S , and controls. UDCA was assessed for its rescue effect on intracellular ATP levels in NM-LRRK2G2019S and in a LRRK2 transgenic fly model with dopaminergic expression of LRRK2 G2019S .Results: Crucial parameters of mitochondrial function were similarly reduced in both M- LRRK2G2019S and NM-LRRK2 G2019S with a specific decrease in respiratory chain complex IV activity. Mitochondrial dysfunction precedes changes in mitochondrial morphology but is normalized after siRNA-mediated knockdown of LRRK2. UDCA improved mitochondrial function in NM-LRRK2 G2019 and rescued the loss of visual function in LRRK2 G2019S flies. Conclusion: There is clear preclinical impairment of mitochondrial function in NM-LRRK2 G2019Sthat is distinct from the mitochondrial impairment observed in parkin-related PD. The beneficial effect of UDCA on mitochondrial function in both NM-LRRK2 G2019S and M-LRRK2 G2019S as well as on the function of dopaminergic neurons expressing LRRK2 G2019S suggests that UDCA is a promising drug for future neuroprotective trials. G2019S 5 manifesting LRRK2 G2019S carriers; NM-LRRK2 G2019S 5 nonmanifesting LRRK2 G2019S carriers; PD 5 Parkinson disease; SSVEP 5 steady-state visual evoked potentials; TUDCA 5 taurine conjugate; UDCA 5 ursodeoxycholic acid.

Our understanding of Parkinson's disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor ‘BMPPB-32’. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK− null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.