Fig 1: Histopathological characterization of the FTLD-TDP case with an R64Gfs*90 TUBA4A mutation. (a–h) pTDP-43 pathology is spread over all layers of the frontal cortex in the R64Gfs*90 TUBA4A mutation case, whereas in a typical FTLD-TDP type C case predominantly the second layer is affected (a,b). pTDP-43 pathology in the R64Gfs*90 TUBA4A mutation case mainly consists of dystrophic neurites of various length and thickness, and few neuronal cytoplasmic inclusions in the frontal cortex (layers II and V depicted; (c–f)). The dentate gyrus shows typical pTDP-43-positive cytoplasmic inclusions (g,h). Scale bars represent 100 μm (a,b) and 50 μm (c–h).
Fig 2: Pedigree of the family of the FTD patient with an R64Gfs*90 TUBA4A mutation. Diamond-shaped symbols were used for anonymity. Filled black symbols represent clinically affected patients. A diagonal line marks deceased patients. The individual ID and relevant clinical neurological diagnosis are mentioned for each patient. The FTD patient with an R64Gfs*90 TUBA4A mutation is indicated with a red arrow. PD = Parkinson’s disease; MSA = multi system atrophy; SD = semantic dementia; EPS = extrapyramidal symptoms.
Fig 3: Imaging of the FTD patient with an R64Gfs*90 TUBA4A mutation. (a,b) Stereotactic surface projections showing lateral views of the left (a) and right (b) hemisphere of an 18F-FDG-PET of the patient, with areas of significant decrease of glucose metabolism superimposed. The scan shows a regional decrease in glucose metabolism in the anterior temporal lobes, more pronounced to the right compared to the left. Color-coding refers to Z-scores with respect to a dataset of normal control subjects. (c,d) MRI (T2-weighted) at the level of the temporal lobe (coronal in (c); horizontal in (d)) shows temporal lobar degeneration typical for FTLD. L = left; R = right.
Fig 4: R64Gfs*90 TUBA4A mutation does not give rise to a mutant protein fragment. (a) Schematic overview of the TUBA4A protein structure indicating the location of the R64Gfs*90 and W407* mutations. The asterisk indicates the location of the early stop codon after amino acid 154 due to the R64Gfs*90 frameshift. The predicted molecular weight of the protein fragment is 16.8 kDa. (b) Western blot on the total fraction of the frontal cortex of the R64Gfs*90 TUBA4A mutation case using an antibody directed against the N-terminal part of TUBA4A, indicating that there was no truncated TUBA4A protein product present. (c) Western blot using an anti-HA-tag antibody on zebrafish lysates at 6 h post fertilization after the injection of wild-type, R64Gfs*90 or W407* mutant TUBA4A mRNA. No R64Gfs*90 TUBA4A protein fragment could be detected around 16.8 kD (asterisk), while the W407* shortened protein product was present. N = N-terminus; C = C-terminus.
Fig 5: R64Gfs*90 TUBA4A mutation causes reduction in levels of wild-type TUBA4A protein. (a) Biochemical analysis of the total TUBA4A expression levels in control cases (F = frontal, P = precentral cortex), FTLD-TDP patients (F = frontal cortex) and different brain regions of the R64Gfs*90 TUBA4A mutation case (frontal cortex, temporal cortex, precentral cortex and cerebellum). (b) Quantification relative to GAPDH shows decreased TUBA4A protein expression in affected brain regions in the R64Gfs*90 TUBA4A mutation case. Each data point represents a single patient.
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