In vivo cellular screening methods and compositions for modeling and treating nervous system dysfunction
Described herein are methods and composition for identifying agents that modulate nerve regeneration in vivo in extended third instar (ETI) Drosophila larvae. The methods include the use of ETI Drosophila larvae having a structural or functional disruption in one or more neurons (e.g., motor neurons) to evaluate a nerve regeneration phenotype over an extended developmental time period in the presence or absence of a test agent.
1. A method for identifying an agent that modulates motor or sensory neuron axon regeneration in an extended third instar (ETI) Drosophila larva, comprising:
(i)contacting with a test agent an ETI Drosophila larva comprising a structural or functional disruption of one or more motor or sensory neuron axons;
(ii) assessing motor or sensory neuron axon regeneration in the disrupted motor or sensory neuron axons by assessing one or more of growth of the disrupted motor or sensory neuron axons, neuromuscular junction formation, glial activation, motor or sensory neuron survival, or neuromuscular junction loss in the contacted ETI Drosophila larva, and
(iii) identifying the test agent as an agent that modulates motor or sensory neuron axon regeneration if a result of the assessment of step (ii) in the presence of test agent differs from the result of the assessment in the absence of the test agent.
2. The method of claim 1 , wherein the ETI Drosophila larva comprises a genetic modification that reduces expression of torso in the prothoracic gland relative to expression of torso in the prothoracic gland of a Drosophila that does not comprise the genetic modification.
3. The method of claim 2 , wherein the genetic modification comprises a transgene to express torso RNAi.
4. The method of claim 3 , wherein the torso RNAi is expressed selectively in the prothoracic gland.
5. The method of claim 4 , wherein the genetic modification comprises a phm-Gal4 transgene and a UAS-torso RNAi transgene.
6. The method of claim 1 , wherein the ETI Drosophila larva comprises a genetic modification to reduce expression of Smad2 in the prothoracic gland.
7. The method of claim 6 , wherein the genetic modification comprises a phm-Gal4 transgene and a UAS-Smad2 RNAi transgene.
8. The method of claim 1 , wherein the ETI Drosophila larva comprises a phm-Gal4 transgene and a UAS promoter driving expression of an RNAi against ras85D RNAi, or ERK RNAi.
9. The method of claim 1 , wherein the contacting step is performed at least about 144 hours after the egg for the ETI Drosophila larva is laid.
10. The method of claim 1 , wherein the contacted ETI Drosophila larva comprises a nerve pinch injury to the one or more motor neurons.
11. The method of claim 1 , wherein the contacted ETI Drosophila larva comprises a genetic modification that induces the structural or functional disruption of the one or more motor neurons.
12. The method of claim 11 , wherein the genetic modification results in expression of at least one heterologous polypeptide associated with a neurodegenerative disease.
13. The method of claim 12 , wherein the at least one heterologous polypeptide comprises a hAPP, hAbeta 1-42 , a hTau, a hsynuclein, hhuntingtin, a hTDP-43, a hSOD, hLRRK2, a hGSK3β, or any combination thereof.