Rapid in vivo forward genetic approach for identifying axon death genes in Drosophila

Abstract

Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFPlabeled, homozygous mutant) on all major autosomal arms (∼95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.neurodegeneration | glial response W idespread axonal degeneration and synapse loss occurs during neurodegenerative disease and after neural trauma. These degradative events result in disruption of neural circuit connectivity and ultimately functional impairment of the nervous system. Identifying molecular cascades that actively promote axonal self-destruction is a key goal. However, despite decades of work, remarkably little is known about the molecular pathways that drive the degeneration of neurites or synapses in any context (1, 2).Axotomy-induced axon degeneration (termed Wallerian degeneration, WD) serves as a useful model to study the mechanisms of axonal self-destruction. When axons are severed, the portion of the axon distal to the injury site and its synapses undergo catastrophic fragmentation after a defined latent phase, and the resulting debris is eventually cleared by surrounding glial cells. The discovery of the spontaneous Wallerian degeneration slow (Wld S ) mouse revealed, surprisingly, that severed axons can in fact survive for weeks in the absence of a cell body (3). It also led to the proposal that "axon death" signaling cascades might exist, akin to apoptotic cell death programs, which actively drive the destruction of the axon (4).Interestingly, Wld S provides significant suppression in mouse models of progressive motor neuron disease and glaucoma (5-8), and moderate protection from chemotherapy-induced axon degeneration (9). These observations argue that defining the molecular mechanisms of axon degeneration in the context of WD could have an important therapeutic impact on the treat...

Age-Dependent TDP-43-Mediated Motor Neuron Degeneration Requires GSK3, hat-trick, and xmas-2

“…We then used mosaic analysis with a repressible cell marker (MARCM) [13], which through its sparse labeling facilitated detailed examination of subcompartments of individual motor and sensory neurons (Figure 1A,B) [14]. To induce MARCM leg clones we tested a number of genetically-encoded Flippase (FLP) sources we recently developed [15]. We found that by driving FLP with the promoter from the proneural gene asense ( ase-FLP ) we could efficiently induce leg MN clones in ~90% of animals.…”

“…To test this hypothesis we severed axons in the adult wing [15] to determine if Sgg J11 , Hat-trick 71 and Xmas-2 45 could suppress WD. However, we found that none of these mutants significantly delayed WD (Figure S3J,K).…”

Using Drosophila models of Huntington's disease as a translatable tool

“…However using standard approaches loss-of-function mutations, which cause lethality, but may otherwise change a mutant Htt induced phenotype, would not be found. To overcome this, potentially lethal genes can be can be knocked out clonally, in an otherwise heterozygous background (Lee and Luo, 2001;Neukomm et al, 2014).…”

“…Using clonal systems also enables the selective labeling of neurons that can be visualized at single axon resolution (Lee and Luo, 2001;Neukomm et al, 2014). M a n u s c r i p t…”

Neurodegeneration: A Leg Up on TDP-43

“…When combined with GAL4 driver lines, transgene expression can be limited to MARCM clones within very few neurons of a subtype. This allowed them to discriminate the morphology of single axons in the fly wing in an assay for injuryinduced axonal degeneration [10]. Here, they report a similar MARCM approach that allows for the expression of human TDP-43 in only a few motor or sensory neurons in the fly leg [4].…”