Song Laboratory Research Overview

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown.

The Song Lab team published findings that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the central nervous system (CNS), and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal.

Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. This group's study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. This work established an important new model system—the fly da neuron regeneration model that resembles the mammalian injury model—with which to study and gain novel insights into the regeneration machinery.

Related Publications

Song Y, Ori-McKenney KM, Zheng Y, Han C, Jan LY, Jan YN. Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam. Genes Dev. 2012 Jul 15;26(14): 1612-1625. PMID: 22759636

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. This group previously reported that the Rtca-mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress; however, the downstream effectors remain unknown.

Through transcriptome profiling, this research show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, these findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Related Publications

Monahan Vargas, EJ, Matamoros AJ, Qiu J, Jan CH, Qin Wang, Gorczyca D, Han TW, Weissman JS, Jan YN, Banerjee S, Song Y. The microtubule regulator ringer functions downstream of the RNA repair/splicing pathway to promote axon regeneration. Genes Dev. 2020 Feb 1;34(3-4):194-208. PMID: 31919191

Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. In this investigation, the team utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. The finding showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system, but also allowed temporal tuning and proper guidance of axon regrowth.

Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. Non-neuronal optogenetics targets damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

Related Publications

Wang Q, Fan H, Li F, Sharum SR, Krishnamurthy V, Song Y, Zhang K. Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in Drosophila. eLife. 2020 Oct 6;9:e57395. PMID: 33021199

Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. The Song Lab team investigated this inhibitory milieu, showing that it is reversible and dependent on glial metabolic status.

Findings show that glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically and pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration.

L-lactate and L-2HG from glia acted on neuronal metabotropic GABAB receptors to boost cAMP signaling. Applying L-lactates to an injured spinal cord promoted corticospinal tract axon regeneration and lead to behavioral recovery in an animal model. These findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.

Related Publications

Li F, et al. Glial metabolic reprogramming promotes axon regeneration and functional recovery in the central nervous system. Cell Metab. 2020 Nov 3;767-785.e7. PMID: 32941799

Cells sense and respond to mechanical stimuli by converting those stimuli into biological signals, a process known as mechanotransduction. Mechanotransduction is essential in diverse cellular functions, including tissue development, touch sensitivity, pain, and neuronal pathfinding.

In the search for key players of mechanotransduction, several families of ion channels were identified as being mechanosensitive and were demonstrated to be activated directly by mechanical forces in both the membrane bilayer and the cytoskeleton. More recently, Piezo ion channels were discovered as a bona fide mechanosensitive ion channel, and its characterization led to a cascade of research that revealed the diverse functions of Piezo proteins and, in particular, their involvement in neuronal repair.

Neurons exhibit a limited ability of repair. Given that mechanical forces affect neuronal outgrowth, it is important to investigate whether mechanosensitive ion channels may regulate axon regeneration. The Song team's research shows that Piezo, a Ca²⁺-permeable nonselective cation channel, functions as an intrinsic inhibitor for axon regeneration in Drosophila. Piezo activation during axon regeneration induces local Ca²⁺ transients at the growth cone, leading to activation of nitric oxide synthase and the downstream cGMP kinase foraging or PKG to restrict axon regrowth.

Loss of Piezo enhances axon regeneration of sensory neurons in the peripheral and central nervous system. Conditional knockout of its mammalian homolog Piezo1 in vivo accelerates regeneration, while its pharmacological activation in vitro modestly reduces regeneration, suggesting the role of Piezo in inhibiting regeneration may be evolutionarily conserved. These findings provide a precedent for the involvement of mechanosensitive channels in axon regeneration and add a potential target for modulating nervous system repair.

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. This group's research shows that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25, promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown, impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury.

Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream nitric oxide signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. The Song Lab's findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.

Related Publications

Song Y, et al. The mechanosensitive ion channel Piezo inhibits axon regeneration. Neuron. 2019 April 17;102(2):373-389.e6. PMID: 30819546

Li F, et al. The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation. Nat Commun. 2021 Jun 22;12(1):3845. PMID: 34158506

Li F, Song Y. Non-canonical role of the ATR pathway in axon regeneration as a mechanosensitive brake. Neural Regen Res. 2022 Nov;17(11): 2423-2324

Miles L, Powell J, Kozak C, Song Y. Mechanosensitive Ion Channels, Axonal Growth, and Regeneration. Neuroscientist. 2022 Apr 13:10738584221088575. PMID: 35414308.

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience, and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, the Song Lab team established a behavioral assay in Drosophila melanogaster and investigated the relationship between sexual experience and aggression.

In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel ppk29 and was mediated by male-specific GABAergic neurons acting on the GABA_A receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. Based on the finding, the researchers propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression.

Related Publications

Yuan Q, Song Y, Yang C, Jan LY, Jan YN. Female exposure modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila. Nat Neurosci. 2014 Jan;17(1): 81-88. PMID: 24241395