TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6

Abstract

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.

Keywords: ALS; Drosophila; Glypican; Motor neuron; Neuromuscular junction; TDP-43; Translation; Wnt signaling.

Fig. 1

mRNAs Enriched with TDP-43 in A Drosophila Model of ALS. a Experimental schematic for RNA immunoprecipitations of human TDP-43, specifically from the motor neurons of third instar larvae. b, c Volcano plot displaying mRNAs enriched with TDP-43 in TDP-43^WT (b) and TDP-43^G298S. All genes associated with TDP-43 (log2FC > 0) are displayed regardless of significance. c) proteinopathy, relative to transcript levels in the ventral nerve cord. d–g GO terms for TDP-43 enriched genes (log2FC > 2, P_adj < 0.05) either unique to TDP-43^WT (e) or TDP-43^G298S (f) or shared between genotypes (g)

Fig. 2

Translational Alterations Induced by TDP-43 Proteinopathy. a Experimental schematic for RNA immunoprecipitations of human TDP-43, specifically from the motor neurons of third instar larvae. b STRING clusters from genes in the top 10% of normalized counts associated with RpL10-GFP in w¹¹¹⁸ larvae (control motor neuron translatomes). c Altered ribosome association of spliceosome genes in TDP-43^WT relative to the control. d Altered ribosome association of purine metabolism genes in TDP-43^G298S relative to control. e Altered ribosome association of oxidative phosphorylation genes in TDP-43^G298S relative to control. f Altered ribosome association of translation associated genes in TDP-43^G298S relative to control. g, h GO terms for RpL10 depleted genes (log2FC < −1) (g) and RpL10 enriched genes (log2FC > 1) (h) shared between both TDP-43^WT and TDP-43^G298S models

Fig. 3

Direct Targets of TDP-43 Mediated Translation Regulation. a Overlap between genes enriched with TDP-43 (log2FC > 1, P_adj < 0.05) and depleted with RpL10 (log2FC < −1) relative to the w¹¹¹⁸ control. b, c GO terms for TDP-43 enriched, RpL10 depleted genes.in TDP-43^WT (b) or TDP-43^G298S (c) models. d, e RpL10 depletion (TRAP) and TDP-43 enrichment (RIP) for genes constituting significant GO terms in TDP-43^WT (d) and TDP-43^G298S (e). f Overlap between genes enriched with TDP-43 (log2FC > 1, P_adj < 0.05) and enriched with RpL10 (log2FC > 1) relative to the w¹¹¹⁸ control. g, h GO terms for TDP-43 enriched, RpL10 enriched genes.in TDP-43^WT (g) or TDP-43^G298S (h) models. i, j RpL10 enrichment (TRAP) and TDP-43 enrichment (RIP) for genes constituting significant GO terms in TDP-43^WT (i) and TDP-43^G298S (j). d–e, i–j Closest human orthologs identified using DIOPT are denoted in parentheses to the right of the fly gene names

Fig. 4

dlp mRNA, a Wg/Wnt interactor, is enriched in TDP-43 complexes and sequestered in insoluble fractions. a STRING diagram [102] depicting relationship between Wg/Wnt signaling genes in the top third of the TRAP control IP normalized counts (D42 > RpL10 GFP, see Materials and Methods). Line thickness between genes is correlated with the strength of the interaction as defined by STRING. b Altered ribosome association of a subset of Wnt signaling genes in TDP-43^G298S relative to control. c qPCR quantification of dlp mRNA enrichment with TDP-43 complexes in TDP-43^WT and TDP-43^G298S expressing larvae (D42 > TDP-43 WT or G298S). Enrichment in TDP-43 complexes is shown as arbitrary units (a.u.) after normalization to input. d,e. Schematic of fractionation experiments and qPCR quantification of dlp mRNA levels in the insoluble fraction (Urea fraction) of TDP-43^WT or TDP-43^G298S expressing larvae relative to w¹¹¹⁸ controls. N = (w¹¹¹⁸ = 5, other = 6) (d), and CRISPR-TDP-43^G294A (relative to CRISPR-TDP-43^WT, N = 3). Significance was determined using the Holm Sidak’s multiple comparisons test (b, d) or Student’s T Test (e). *P_value < 0.05, ***P_value < 0.001. Error bars represent SEM

Fig. 5

Dlp protein expression is altered by TDP-43 proteinopathy. a–l Representative images of w¹¹¹⁸, TDP-43^WT, TDP-43^G298S and TBPH^RNAi NMJs stained for Dlp and HRP. m Quantification of Dlp protein relative to bouton area in terminal boutons. N = 13 for w¹¹¹⁸, 15 for TDP-43^WT, 16 for TDP-43^G298S, 6 for TBPH^RNAi. n–y Representative images of w¹¹¹⁸, TDP-43^WT, TDP-43^G298S and TBPH^RNAi VNCs stained for Dlp, DNA and TDP-43 (z). Quantification of Dlp granule number in the neuropil. N = 10 for w¹¹¹⁸, 10 for TDP-43^WT, 9 for TDP-43^G298S, 6 for TBPH^RNAi. Genotypes and stainings, as indicated. Scale bars: a 10 μm, n 50 μm. v 22 μm. Significance determined using the Holm Sidak’s multiple comparison test. *P_value < 0.05, **P_value < 0.01, ***P_value < 0.001. Error bars represent SEM

Fig. 6

Altered dlp mRNA levels modify TDP-43 induced locomotion defects. a Larval turning times for dlp mRNA overexpression in the w¹¹¹⁸ genetic background (control for OE experiments), TDP-43^WT, TDP-43^G298S and TBPH^RNAi. N = 29 for w¹¹¹⁸, 29 for dlp^OE, 30 for TDP-43^WT, 37 for TDP-43^WT dlp^OE, 31 for TDP-43^G298S, 36 for TDP-43^G298S dlp^OE, 32 for TBPH^RNAI, 31 for TBPH^RNAi dlp^OE. b Larval turning times for dlp RNAi in the attp40 genetic background (control for RNAi experiments), TDP-43^WT, and TDP-43^G298S. N = 30 for attp40, 31 for dlp^RNAi, 29 for attp40 TDP-43^WT, 37 for TDP-43^WT dlp^RNAi, 29 for attp40 TDP-43^G298S, 38 for TDP-43^G298S dlp^RNAi, 30 for TBPH^RNAi, 28 for TBPH^RNAi dlp^RNAi. Significance determined using the MannU Whitney Test or Holm Sidak’s multiple comparison test. *P_value < 0.05, **P_value < 0.01, ***P_value < 0.001. c–n Representative images of dlp^OE NMJs in the context of w¹¹¹⁸, TDP-43^WT, TDP-43^G298S and TBPH^RNAi stained for Dlp and HRP. o Quantification of Dlp intensity in NMJ terminal boutons. N = 5 for w¹¹¹⁸ dlp^OE, 5 for TDP-43^WT dlp^OE, 5 for TDP-43^G298S dlp^OE, 6 for TBPH^RNAi dlp^OE. Genotypes and stainings, as indicated. Scale bars: c 10 μm. *P_value < 0.05, **P_value < 0.01, ***P_value < 0.001. Error bars represent SEM

Fig. 7

GPC6, a Dlp ortholog exhibits increased expression and altered localization in patient spinal tissues. a Representative image of a control spinal cord (case number CW01-72). N = 7. b Representative image of ALS patient spinal cord. (GWF15-07). N = 16. c Quantification of GPC6 granule number in post-mortem spinal tissue. Arrowheads indicate motor neurons. Significance determined using the Mann U Whitney Test. *P_value < 0.05, Scale bar in a 40 μm. Error bars represent SEM

Fig. 8

Dlp is altered in the context of TDP-43 proteinopathy. Dlp mRNA is enriched in TDP-43 complexes, depleted from ribosomes and sequestered in insoluble/urea complexes. Taken together, these findings and our observations of Dlp protein being reduced at the NMJ and accumulating in puncta within cell bodies and neuropils suggest multiple cellular defects including local translation inhibition at synapses, axonal trafficking and endomembrane trafficking deficits