TBK1 haploinsufficiency results in changes in the K63-ubiquitination profiles in brain and fibroblasts from affected and presymptomatic mutation carriers

Abstract

Background: Frontotemporal dementia (FTD) is a neurodegenerative disease, resulting in progressive problems in language and/or behaviour and is often diagnosed before 65 years of age. Ubiquitin positive protein aggregates in the brain are among the key pathologic hallmarks of frontotemporal lobar degeneration (FTLD) postmortem. The TANK-binding kinase 1 gene (TBK1) is on the list of genes that can contribute to the development of FTD as well as the related neurodegenerative disease amyotrophic lateral sclerosis (ALS).

Methods: In this study, using an array of clinical and neuropathological data combined with biochemical and proteomics assays, we analyze the TBK1 splice-mutation (c.1340 + 1G > A) in a Swedish family with a history of FTD and ALS. We also explore the K63 ubiquitination landscape in post-mortem brain tissue and fibroblast cultures.

Results: The intronic (c.1340 + 1G > A) mutation in TBK1 results in haploinsufficiency and affects the activity of the protein in symptomatic and pre-symptomatic mutation carriers.

Conclusion: Our results suggest that the mutation leads to a significant reduction of TBK1 activity and induce alterations in K63 ubiquitination profile of the cell already in the presymptomatic stages.

Keywords: ALS; Autophagy; FTD; FTLD; Frontotemporal dementia; Haploinsufficiency; Neurodegeneration; TBK1; Ubiquitination.

Fig. 1

A Pseudonymized pedigree of the Swedish family with a history of hereditary FTD and ALS caused by the TBK1 p.Ala417* mutation. B Table of demographic data for generations II–IV. C Representation of the number of individuals and the sample types used in the study as well as the genotype and phenotype of the donors. CVL, cerebrovascular lesion; bvFTD, behaviour variant frontotemporal dementia; MCI, mild cognitive impairment; NOS, not otherwise specified; PNFA, progressive non-fluent aphasia; ALS, amyotrophic lateral sclerosis; NA, not available. Marked by (a) indicate the individuals with available brain tissue. b Represent the individuals with confirmed mutation status. c Indicate the individuals with available DNA sample. d Indicate that subject III:7 is known to be a TBK1 non-carrier

Fig. 2

Representative images of the neuropathological examination of TBK1 p.Ala417*-mutation carriers in the family in Figure A. A, B Superior view and coronal section of the brain. There is minimal amount of atrophy which is highlighted with the red box corresponding to the right temporal lobe. C Hematoxylin and eosin staining of frontal cortex with some superficial vacuolization. D p62 staining of frontal cortex. E) Ubiquitin staining of frontal cortex. F pTDP-43 staining of the granular cells of the dentate gyrus in hippocampus G pTDP-43 staining of frontal cortex. H pTDP-43 staining of the white matter. The scale bars represent 50 µm. Pictures are taken from three of the four postmortem cases: Panels A and B from one individual (diagnosed with Dementia-NOS), panels C, F, G and H are from a second individual (diagnosed with Dementia-NOS frontal) and panels D, and E are from a third individual with bvFTD

Fig. 3

A Forward and reverse primers used in the TBK1 transcript analyses illustrated in panels B–G. The amplicon contains the location of the TBK1 c.1340 + 1G > A (p.Ala417*) mutation in intron 11. B Image of an agarose gel electrophoresis of cDNA extracted from blood in non-carriers (lanes 1, 3, 4, and 5) and a presymptomatic mutation carrier (lane 2). The asterisk indicates the wildtype transcript and the arrowhead indicates the shorter mutated transcript with an exon-11 deletion. C–G ddPCR of cDNA from blood (two presymptomatic mutation carriers and seven non-carriers), fibroblasts (one presymptomatic mutation carrier and one non-carrier) and frozen brain tissue (one mutation carrier with FTD and two non-carriers). Clusters are formed from the signals in single droplets: blue (wildtype transcript), green (mutated transcript) and grey (empty droplets). C, D Representative two-dimensional ddPCR plots. E–G Absolute number of transcript copies per microliter. Statistical differences were calculated by one-way ANOVA followed by Tukey’s Multiple comparison post hoc Test. Adjusted p values: *p < 0.020, **p < 0.0007, ***p < 0.005. Taken together our results confirm previously described haploinsufficiency of TBK1 in affected mutation-carriers and we also show that the haploinsufficiency is present already at the presymptomatic stage

Fig. 4

Western blot analysis of TBK1 expression; A Expression of TBK1 and pTBK1 is reduced in the brain lysates from an affected mutation carrier sample; B Expression of TBK1 and pTBK1 is reduced in the fibroblast sample from TBK1 pre-symptomatic mutation carrier. Quantification of the band intensity relative to tubulin blot and normalized against the non-carrier sample is presented for each panel

Fig. 5

Analysis of the K63 ubiquitination landscape of TBK1 mutation carriers and controls in fibroblasts and brain. A, B Heatmap representing the log2 fold-change values of differentially K63-ubiquitinated proteins in TBK1 mutation carrier samples vs. control samples. Proteins and samples are separately clustered via hierarchical clustering. Proteins are colored based on the intensity of detected signal post TUBE-purification with green representing a fourfold decrease and red representing a fourfold increase in the detected signal relative to the mean signal of the protein across all replicates of each genotype. C, D Volcano plots of proteins in brain (C) and fibroblasts (D) identified by mass spectrometry according to their Benjamini–Hochberg adjusted p value (y axis) and their relative abundance ratio (log2 fold change) (x axis). Red dots represent the proteins detected with increased levels in mutation carrier samples and green dots represent the proteins detected in higher levels in control samples. Orange dots represent proteins that do not fulfill the cutoff of log2 fold difference. Dotted lines present the p value cutoff of p < 0.05 and log2 fold difference