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

Abstract

The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. We used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. We show that expression of TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. We report the identification of three genes that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.

Figure 1. MARCM clones of leg motor neurons can be induced using asense-FLP, and demonstrate progressive NMJ and axon degeneration with overexpression of TDP-43^Q331K

(A) Labeling of all glutamatergic neurons in a middle leg. Arrowheads indicate sensory cell bodies. Red and yellow boxes indicate the regions used for imaging axons and NMJs respectively for all subsequent quantification. (B) Low power image of a single motor neuron clone. Magnification of a nerve terminal (inset) highlights mCD8::GFP punctae. (C) Colocalization of mCD8::GFP with mCherry tagged active zone protein Bruchpilot (Schmid et al., 2008). (D) 3D projection view of a leg NMJ demonstrates close apposition of post-synaptic GluRIIA-mRFP and pre-synaptic mCD8::GFP. (E) Time course of motor axon and NMJ degeneration caused by TDP-43^Q331K. (F) Quantification of mild to moderate axon degeneration, comparing leg motor with wing sensory neurons. Genotypes used: (A) w ; OK371-Gal4, UAS-mCD8:: GFP; UAS-mCD8::GFP (B) w, tub-Gal80, FRT19A/FRT19A ; OK371-Gal4, UAS-mCD8:: GFP ; UAS-mCD8::GFP, ase-FLP (C) w, tub-Gal80, FRT19A/FRT19A ; OK371-Gal4, UAS-mCD8::GFP, UAS-Brp^mCh; UAS-mCD8::GFP, ase-FLP (D) w, tub-Gal80, FRT19A/FRT19A ; OK371-Gal4, UAS-mCD8::GFP ; UAS-mCD8::GFP, ase-FLP/dGluRIIA-mRFP (E) w, tub-Gal80, FRT19A/FRT19A ; OK371-Gal4, UAS-mCD8::GFP; UAS-mCD8::GFP, UAS-TDP-43^Q331K, ase-FLP. Scale bars 10µm. See also Figure S1.

Figure 2. Loss of Sgg suppresses TDP-43 toxicity

(A) Motor axon and NMJ morphology of, from left to right: GFP control, TDP-43 control, TDP-43 in line 11 background, GFP in line 11 background. Scale bar 5µm. (B) Quantification of axon degeneration and aggregation of GFP at NMJs in Sgg mutants and rescue crosses with a duplication covering Sgg (stock DC048) and UAS-Sgg. (C) Cartoon of the Sgg gene with the position of the premature stop mutation found in line 11 (L245*). The position of the previously uncharacterized P-element insertion (line sgg^G0183) in the 5’UTR of an alternative transcript is shown for comparison. (D) Western blotting of third instar larval brains from homozygous Sgg mutants and quantification of TDP-43 band intensity. ns, non-significant. See also Figures S2 and S3.

Figure 3. Loss of function mutations in Hat-trick and Xmas-2 suppress TDP-43^Q331K mediated motor neuron degeneration

(A) Motor axon and NMJ morphology of, from left to right: GFP control, TDP-43 control, TDP-43 in line 71 background, TDP-43 in line 45 background. Scale bar 5µm. (B) Quantification of active zones in GFP and TDP-43 background. **** p<0.0001. (C) Quantification of axon and NMJ degeneration in Hat-trick and Xmas-2 mutants, and in rescue experiments with duplication stocks DC353 and DC325. (D) The Hat-trick gene is shown with newly isolated alleles: red arrows indicate premature stop mutations in line 71 (L982*) and 47 (Q2145*), green arrow indicates the intronic donor splice site mutation in line 39 (IVS2+2T>A). (E) Agarose gel electrophoresis of PCR products from cDNA extracted from third instar larval brains of homozygous Hat-trick mutant lines. Location of PCR primers indicated in (D). (F) The Xmas-2 gene is shown with the line 45 premature stop mutation (Q241*, red arrow), and the PBac insertion (blue arrow). (G) Western blotting for Xmas-2 in third instar larval brains from homozygous Xmas-2 mutants. (H) Immunohistochemistry for TDP-43 of thoracic ganglia motor neuron cell bodies at 7dpe. Scale bar 10µm. (I) Western blotting for TDP-43 and GFP of third instar larval brains. Note that the molecular weight of GFP is at 50kDa as it is bound to mCD8 protein. (J) Third instar larval brain quantitative RT-PCR. Homozygous animals of each genotype were used. Expression normalized to that of control brains. Values are the average of three biological replicates and given as mean ± SEM. ns non-significant. See also Figures S2 and S3.