Identification of GNPDA2 molecular interactors. (A) An immunoprecipitation (IP) assay was performed in protein extracts from a human olfactory cell line. ERK antibody and recombinant GNPDA2 (6xHis-tagged) were used as assay controls (left). Metascape analysis indicating the biological pathways governed by GNPDA2 interactors (right). (B) GO analysis showing cellular localization of specific GNPDA2 interactors. (C) Venn diagram showing common GNPDA2, APP and Tau interactors.

RNA-seq analysis in hNECs. (A) Brief description of experimental design. (B) Cell proliferation monitoring (via an xCELLigence analysis, Agilent, SC, USA) and the corresponding Volcano plots of differentially expressed genes (in red) across biological conditions: effect of GNPDA2 overexpression in hNECs, effect of Aβ treatment after GNPDA2 overexpression in hNECs and effect of Tau P301L treatment after GNPDA2 overexpression in hNECs. (C) Overlap across differential RNA-seq datasets. (D) Top 20 functional analyses across the three biological conditions generated by Metascape.

Gene interactome maps for differentially expressed genes in hNECs. Representation of the interactions between differentially expressed genes after GNPDA2 overexpression (A), GNPDA2 overexpression and Aβ treatment (B) and GNPDA2 and Tau overexpression (C). The color of each gene represents its up (red) and down (green) expression found in the RNAseq experiment. White nodes represent targets potentially responsible for the deregulation of the surrounding genes.

Gene interactome maps for differentially expressed genes in hNECs. Representation of the interactions between differentially expressed genes after GNPDA2 overexpression (A), GNPDA2 overexpression and Aβ treatment (B) and GNPDA2 and Tau overexpression (C). The color of each gene represents its up (red) and down (green) expression found in the RNAseq experiment. White nodes represent targets potentially responsible for the deregulation of the surrounding genes.

Study of GNPDA2 overexpression in zf Tau P301L transgenic embryos. (A) Experimental design used in the zebrafish model. (B) Representative pictures of 30 hpf not-injected embryos or embryos injected with cLuc and GNPDA2 mRNAs (both DsRed/mutant TAU-positive and -negative embryos) after whole-mount immunostaining with znp1/zn-1 antibodies. White asterisks indicate the position of the four axonal extensions quantified in not injected Tau− pictures. (C) Graph shows the mean ± SEM of the total axon length of the first four caudal primary motoneurons anterior to the end of the yolk extension of the indicated experimental groups. ** p < 0.01; *** p < 0.001. (D) Representative pictures of 48 hpf not-injected embryos or embryos injected with cLuc and GNPDA2 RNAs (both DsRed/mutant TAU-positive and -negative embryos) after TUNEL staining. The spinal cord area, where the number of TUNEL-positive cells was quantified, is highlighted in not injected Tau− pictures. (E) Graph shows the mean ± SEM of the number of TUNEL-positive neurons present along the spinal cord area of the indicated experimental groups. *** p < 0.001. (F) Western blot developed with anti-Tau (Phospho S396), anti-Tau (total) and anti-GNPDA2 antibodies of 48 hpf embryos from the indicated experimental groups. Ponceau S red staining corresponding to fragments used for developing pTau and total Tau are shown below each antibody-specific staining. Numbers correspond to the optical density quantification (arbitrary units) of Ser396-phosphorylated Tau with respect to the total Tau levels. Specimens in the study: not injected Tau− (zebrafish specimens with no Tau and no GNPDA2); not injected Tau+ (zebrafish specimens overexpressing human TauP301L and no GNPDA2); cLUC Tau− (zebrafish specimens with no Tau and expressing the luciferase vector); cLUC Tau− (zebrafish specimens overexpressing human TauP301L and expressing the luciferase vector); GNPDA2 Tau− (zebrafish specimens with no Tau and expressing human GNPDA2) and GNPDA2 Tau+ (zebrafish specimens overexpressing human TauP301L and expressing human GNPDA2).

Proteomics in h.Tau P301L zebrafish embryos. (A) Heatmap representation showing differentially expressed proteins (DEPs) between not-injected Tau− and not-injected Tau+. (B) Functional clustering of DEPs between not-injected Tau– and not-injected Tau+ using Metascape. (C) Venn diagram showing DEPs previously described as Tau interactors. (D) Synaptic ontology analysis (subcellular distribution) of DEPs using the SynGo tool.

Differentially expressed proteins across Tau P301L embryos overexpressing GNPDA2. (A) Heatmap representing the differential zebrafish proteome across not-injected Tau−, not-injected Tau+ embryos, cLUC Tau+ and GNPDA2 Tau+ embryos. (B) Protein clusters specifically modulated across the different experimental conditions and functional clustering based on the specific disrupted zebrafish proteomes.

Serum GNPDA2 levels across neurological disorders. (A) GNPDA2 levels measured in the sera derived from 215 individuals with different neurological syndromes. (B) Sex-specific analysis across neurological disorders (71 controls; mean age: 69.2 years; 40M/31F, 40 AD subjects: mean age: 75.1 years; 20M/20F, 40 PSP subjects; mean age: 67.9 years; 15M/25F, 40 DFT subjects; mean age: 69.5 years; 20M/20F, 12 DLB subjects; mean age: 73.5 years; 6M/6F; 12 ALS subjects; mean age: 57.8 years; 5M/7F; ELISA (Mann–Whitney U test; ** p-value < 0.01; *** p-value: 0.001)).