TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization

Abstract

TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previously, we have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions. We have now investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result we found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, we observed that TDP-43 physically interacts with futsch mRNA and that its RNA binding capacity is required to prevent futsch down regulation and synaptic defects.

Figure 1. Loss of TBPH function affects synaptic growth and boutons shape.

(A) Confocal images of wild type NMJs on muscle 6 and 7, abdominal segment II, at L1 (38 h), L2 (62 h) and L3 (110 h) stages using α-HRP antidody. (B) And (C) Similar α-HRP staining showing NMJ morphology at different stages of larval development in TBPH^D23 and TBPH^D142 homozygous larvae. Scale bar 20 µm. (D) Quantifications showing total number of boutons present in the abdominal segment II of wild type and TBPH mutant alleles during larval development. (E) Regular shape and distribution of 1b boutons in wild type NMJs compared to misshapen boutons in (F) TBPH^D23/- (G) TBPH^D142/- and (H) TBPH^D23/+;elav>TBPH-RNAi mutants. (I) Bouton shape is rescued by expressing UAS TBPH in motor neurons with D42-GAL4. Scale bar 5 µm. (J) Quantifications showing regular and irregular boutons present in the abdominal segment II at third instar larval stages in wild type and mutant alleles. *** Indicates p<0.001 calculated by one-way ANOVA. Error bars indicate SEM.

Figure 2. Loss of TBPH interferes with microtubule organization.

(A) Confocal images showing futsch staining at the terminal synaptic boutons of (Ai–Aiii) wild type, (Aiv–Avi) TBPH^D23, (Avii–Aix) TBPH^D142. Note the complete absence of futsch at the most distal, newly formed, boutons in TBPH mutants (arrow head). (Ax–Axii) Expression of TBPH protein by D42-Gal4 rescues futsch staining in TBPH mutant larvae. (B) Quantifications of futsch staining pattern in muscle 6–7 abdominal segment II showing increased number of diffused futsch and futsch negative boutons in TBPH mutant alleles compared to wild type. n = 15 larvae. **p<0.01 and ***p<0.001 calculated by one-way ANOVA. (C) Western blot analysis and the respective histogram confirmed the reduced futsch expression levels in TBPH mutant fly heads compared to wild type. n = 4.

Figure 3. Reduced stable MT and Tubulin acetylation in TBPH mutants.

(A) Confocal images showing wild type boutons with stable MT bundles (Ai–Aiii, arrowhead) labeled by acetylated Tubulin. TBPH mutants (Aiv–Avi) TBPH^D23/-, (Avii–Aix) TBPH^D142/- show the absence of acetylated MTs staining at the distal boutons (arrowhead). (Ax–Axii) D42-GAL4 driven TBPH expression rescues the lack of stable MTs in the TBPH loss of function. Scale bar 5 µm. (B) Quantifications of stable MT labeled by acetylated Tubulin in the abdominal segment II showing significant labeling reduction in TBPH mutant alleles compared to wild type larval NMJ. ***p<0.001 calculated by one-way ANOVA. n = 16 larvae.

Figure 4. TBPH function requires futsch activity to promote NMJ growth.

NMJ morphology in muscle 6/7 abdominal segment II of (A) futsch^N94/+ (B) TBPH^D23/- (C) TBPH^D23/-;elav>TBPH (D) futsch^N94/+;TBPH^D23/-;elav>TBPH (E) TBPH^D23/-;elav>Tau^wt, Scale bar 20 µm is valid for all figures. Note the NMJ growing defects in futsch^N94/+;TBPH^D23/-;elav>TBPH genotypes compared to TBPH^D23/-;elav>TBPH. Human Tau protein expression showed the recovery in NMJ growing defects observed in TBPH mutants. (F) Quantifications showing significant reduction in the number of boutons and (G) the number of branches in futsch^N94/+;TBPH^D23/-;elav>TBPH genotypes compared to TBPH^D23/-;elav>TBPH. Tau protein expression recovered the NMJ growing defects observed in TBPH mutants. n = 13 larvae.

Figure 5. TBPH function requires futsch activity to stabilize presynaptic MTs.

(A) Confocal images showing the distribution of acetylated MTs in distal boutons of (Ai–Aiii) futsch^N94/+, (Aiv–Avi) TBPH^D23/-, (Avii–Aix) TBPH^D23/-;elav>TBPH, (Ax–Axii) futsch^N94/+;TBPH^D23/-;elav>TBPH and (Axiii–Axv) TBPH^D23/-;elav>Tau. Note that Tubulin acetylation and stable MT reduction was noticed in the distal boutons of Futsch^N94/+; TBPH^D23/-;elav>TBPH compared to similar rescue in the TBPH^D23 mutant background alone (arrow head). Expression of Tau protein in TBPH mutant alleles was able to rescue the distribution of acetylated MTs at synaptic boutons. Scale bar 5 µm valid for all figures. (B) Quantifications of acetylated stable MTs in muscle 6/7 abdominal segments II n = 13 larvae. * p<0.05, ** p<0.01 and ***p<0.001.

Figure 6. TBPH binding to futsch mRNA is required for NMJ growth and MT organization.

(A) qPCR analysis of mRNAs immunoprecipitated by Flag-tagged TBPH. The enrichment-fold is referred to an unrelated protein (REP, Right charts) or to the mutant TBPH^F/L150–152 (FL, Left charts). Significant levels of enrichment were observed for futsch mRNA, together with hdac-6 mRNA and the (UG)9 RNA used as positive controls. The mRNAs of the ribosomal protein rpl-52 and homer were not enriched significantively. (B) Wild type TBPH protein expression in TBPH^D23/-;D42>TBPH flies succeed to rescue the synaptic growth compared to (C) TBPH RRM1 mutant isoform in TBPH^D23/-;D42>TBPH^F/L150–152 larval NMJ, Scale bar 20 µm. (D) Quantifications of big synaptic boutons and (E) terminal branches in muscle 6–7 (abdominal segments II and III) in TBPH^F/L150–152 and TBPH wild type rescues. (F) Quantifications showing the reduced futsch staining in the distal boutons of TBPH^F/L150–152 rescue NMJs compared to wild type TBPH protein. n = 12 larvae. ***p<0.001, **p<0.01. (G) Confocal images showing reduced futsch positive staining in the distal boutons of TBPH^F/L150–152 rescues (Giv-Gvi see arrowhead) compared to wild type TBPH rescues (Gi-Giii). Scale bar 5 µm. (H) Western blot analysis and the respective histogram showing the reduced levels of futsch protein expression in TBPH^D23/-;elav>TBPH^F/L150–152 compared with TBPH^D23/-;elav>TBPH (upper panel) n = 3. Tubulin was used as a loading control (bottom panel).