The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

Abstract

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

Figure 1. Characterization of Cabeza, the Drosophila homolog of human FUS/TLS.

(A) Expression pattern of a caz transgene under the control of the endogenous promoter in the adult brain detected using a FLAG epitope introduced immediately after the start codon. (B) Larval neuronal nuclei expressing genomic Caz detected with FLAG alone (upper panel) or colabeled with the neuronal RNA binding protein Elav. (C) Schematic of caz¹ mutant construction. The transposon EP1564 was mobilized to create a small deletion Df[1]383 that removes 58% of the caz gene, caz promoter sequences, and disrupts the nearby gene CG32576. A rescuing transgene for CG32576 was inserted onto the Df[1]383 chromosome to create caz¹ mutants. (D) Percentage of male larva of the indicated genotypes that eclosed to produce adults (n > 100). Pan-neuronal expression of Caz, human FUS, or ALS mutant FUS (FUS^R522G and FUS^P525L) transgenes rescue eclosion equally (genotype: caz¹, C155-Gal4/Y; UAS transgene). (E) Representative image of 10 superimposed paths of 60 seconds of adult locomotion for control (precise excision) of 1-day-old adult male flies. (F) Representative image of 10 superimposed paths of 60 seconds of adult locomotion of caz¹ mutant 1-day-old adult male flies. (G) Walking speed of 1-day-old adult male flies of the indicated genotypes in a 60-second trial (n > 30). (H) Percentage survival of adult male flies of the indicated genotypes (n > 68). Error bars represent SEM. Scale bars: 100 μm (A); 5 μm (B). *P < 0.05; ***P < 0.001.

Figure 2. caz and tbph are members of a genetic pathway.

(A) Percentage of male larva of the indicated genotypes that eclosed to produce adults (n > 100). The tbph^–/– genotype is tbph^∆23/Df[2R]BSC660. + indicates neuronal expression of UAS-TBPH, UAS-TDP-43, or UAS-Caz with C155-Gal4. (B) Representative images of 10 superimposed paths of 60 seconds of adult locomotion of 1-day-old adult male flies of control, tbph mutant, or caz¹ mutants either alone or expressing UAS-Caz or UAS-TBPH. (C) Walking speed of 1-day-old adult male flies of the indicated genotypes in a 60-second trial (n > 30). (D) Percentage survival of adult male flies of the indicated genotypes (n > 100). Error bars represent SEM. ***P < 0.001.

Figure 3. Caz is epistatic to tbph.

(A–L) Third instar NMJ terminals stained with anti-CSP (green) to label the presynapse and anti-HRP (red) to label the neuronal membrane at muscle 4, segment A3 for motor neuron overexpressing (OE) transgenic Caz, FUS, TBPH, TDP-43, and FUS or TDP-43 ALS mutants in wild-type (B–H) or tbph and caz¹ mutants (J and L) driven in motor neurons by OK6-Gal4. The tbph^–/– genotype is tbph^∆23/Df[2R]BSC660. Overexpression of wild-type TBPH, TDP-43, Caz, or FUS proteins induces NMJ expansion while ALS mutant FUS or TDP-43^M337V does not (B–H). Mutants of caz or tbph have normal NMJ morphology (I and K). The NMJ expansion induced by expression of wild-type TDP-43 is completely suppressed in caz mutants (J); however, the NMJ expansion induced by Caz overexpression is not suppressed in tbph mutants (L). (M) Quantification of synapse terminal bouton number divided by muscle surface area for muscle 4 segment A3 normalized to control. Error bars represent SEM. ***P < 0.001.

Figure 4. Caz and TBPH proteins interact.

(A) YFP-tagged TBPH expressed in Drosophila adult brains with C155-Gal4 coimmunoprecipitates when flag-tagged Caz is used for pulldown. Treatment of brain extracts with RNaseA inhibits this interaction. (B) Endogenous TBPH protein levels were not changed in 1-day-old caz mutant males compared with precise excision controls, and genomic flag-Caz protein is similar in 1-day-old tbph mutants compared with controls. (C) 4 of the 8 C-terminal amino acids of human FUS and Drosophila Caz are identical (green), and 2 (red) were mutated in Caz cDNA transgenes. (D) Drosophila motor neuron cell bodies expressing UAS-Caz, UAS-Caz^R395G, and UAS-Caz^P398L (green), histone-YFP (red), and cytoplasmic β-galactosidase (blue) driven by the motor neuron driver OK319-Gal4 or single channel images of Caz or Caz mutants (gray). Unlike wild-type Caz, both Caz C-terminal mutants are found extensively in the cytoplasm. (E) YFP-TBPH coimmunoprecipitates when flag-tagged Caz^R395G or Caz^P398L is used for pulldown from adult brain extracts.