Voltage-gated calcium channels act upstream of adenylyl cyclase Ac78C to promote timely initiation of dendrite regeneration.

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.

Dendrite regeneration mediates functional recovery after complete dendrite removal.

Unlike many cell types, neurons are not typically replaced if damaged. Therefore, regeneration of damaged cellular domains is critical for maintenance of neuronal function. While axon regeneration has been documented for several hundred years, it has only recently become possible to determine whether neurons respond to dendrite removal with regeneration. Regrowth of dendrite arbors has been documented in invertebrate and vertebrate model systems, but whether it leads to functional restoration of a circuit remains unknown. To test whether dendrite regeneration restores function, we used larval Drosophila nociceptive neurons. Their dendrites detect noxious stimuli to initiate escape behavior. Previous studies of Drosophila sensory neurons have shown that dendrites of single neurons regrow after laser severing. We removed dendrites from 16 neurons per animal to clear most of the dorsal surface of nociceptive innervation. As expected, this reduced aversive responses to noxious touch. Surprisingly, behavior was completely restored 24 â€‹h after injury, at the stage when dendrite regeneration has begun, but the new arbor has only covered a small portion of its former territory. This behavioral recovery required regenerative outgrowth as it was eliminated in a genetic background in which new growth is blocked. We conclude that dendrite regeneration can restore behavior.

The exocyst complex is required for developmental and regenerative neurite growth in vivo.

The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo we used Drosophila sensory neurons as a model system. To bypass early developmental requirements in other cell types, we used neuron-specific RNAi to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, we considered this could be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, we used laser microsurgery to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. We conclude that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement.

The receptor tyrosine kinase Ror is required for dendrite regeneration in Drosophila neurons.

While many regulators of axon regeneration have been identified, very little is known about mechanisms that allow dendrites to regenerate after injury. Using a Drosophila model of dendrite regeneration, we performed a candidate screen of receptor tyrosine kinases (RTKs) and found a requirement for RTK-like orphan receptor (Ror). We confirmed that Ror was required for regeneration in two different neuron types using RNA interference (RNAi) and mutants. Ror was not required for axon regeneration or normal dendrite development, suggesting a specific role in dendrite regeneration. Ror can act as a Wnt coreceptor with frizzleds (fzs) in other contexts, so we tested the involvement of Wnt signaling proteins in dendrite regeneration. We found that knockdown of fz, dishevelled (dsh), Axin, and gilgamesh (gish) also reduced dendrite regeneration. Moreover, Ror was required to position dsh and Axin in dendrites. We recently found that Wnt signaling proteins, including dsh and Axin, localize microtubule nucleation machinery in dendrites. We therefore hypothesized that Ror may act by regulating microtubule nucleation at baseline and during dendrite regeneration. Consistent with this hypothesis, localization of the core nucleation protein γTubulin was reduced in Ror RNAi neurons, and this effect was strongest during dendrite regeneration. In addition, dendrite regeneration was sensitive to partial reduction of γTubulin. We conclude that Ror promotes dendrite regeneration as part of a Wnt signaling pathway that regulates dendritic microtubule nucleation.

Ketogenic and anaplerotic dietary modifications ameliorate seizure activity in Drosophila models of mitochondrial encephalomyopathy and glycolytic enzymopathy.

Seizures are a feature not only of the many forms of epilepsy, but also of global metabolic diseases such as mitochondrial encephalomyopathy (ME) and glycolytic enzymopathy (GE). Modern anti-epileptic drugs (AEDs) are successful in many cases, but some patients are refractory to existing AEDs, which has led to a surge in interest in clinically managed dietary therapy such as the ketogenic diet (KD). This high-fat, low-carbohydrate diet causes a cellular switch from glycolysis to fatty acid oxidation and ketone body generation, with a wide array of downstream effects at the genetic, protein, and metabolite level that may mediate seizure protection. We have recently shown that a Drosophila model of human ME (ATP61) responds robustly to the KD; here, we have investigated the mechanistic importance of the major metabolic consequences of the KD in the context of this bioenergetics disease: ketogenesis, reduction of glycolysis, and anaplerosis. We have found that reduction of glycolysis does not confer seizure protection, but that dietary supplementation with ketone bodies or the anaplerotic lipid triheptanoin, which directly replenishes the citric acid cycle, can mimic the success of the ketogenic diet even in the presence of standard carbohydrate levels. We have also shown that the proper functioning of the citric acid cycle is crucial to the success of the KD in the context of ME. Furthermore, our data reveal that multiple seizure models, in addition to ATP61, are treatable with the ketogenic diet. Importantly, one of these mutants is TPIsugarkill, which models human glycolytic enzymopathy, an incurable metabolic disorder with severe neurological consequences. Overall, these studies reveal widespread success of the KD in Drosophila, further cementing its status as an excellent model for studies of KD treatment and mechanism, and reveal key insights into the therapeutic potential of dietary therapy against neuronal hyperexcitability in epilepsy and metabolic disease.

The ATP-sensitive K channel is seizure protective and required for effective dietary therapy in a model of mitochondrial encephalomyopathy.

Effective therapies are lacking for mitochondrial encephalomyopathies (MEs). MEs are devastating diseases that predominantly affect the energy-demanding tissues of the nervous system and muscle, causing symptoms such as seizures, cardiomyopathy, and neuro- and muscular degeneration. Even common anti-epileptic drugs which are frequently successful in ameliorating seizures in other diseases tend to have a lower success rate in ME, highlighting the need for novel drug targets, especially those that may couple metabolic sensitivity to neuronal excitability. Furthermore, alternative epilepsy therapies such as dietary modification are gaining in clinical popularity but have not been thoroughly studied in ME. Using the Drosophila ATP61 model of ME, we have studied dietary therapy throughout disease progression and found that it is highly effective against the seizures of ME, especially a high fat/ketogenic diet, and that the benefits are dependent upon a functional KATP channel complex. Further experiments with KATP show that it is seizure-protective in this model, and that pharmacological promotion of its open state also ameliorates seizures. These studies represent important steps forward in the development of novel therapies for a class of diseases that is notoriously difficult to treat, and lay the foundation for mechanistic studies of currently existing therapies in the context of metabolic disease.