Search

Search Results

1 results found. Results per page: [ 20 ][ 40 ][ 60 ][ 80 ][ 100 ][ 200 ][ 300 ]

Sort by: [ Publication Date ][ Score ]

Year of publication: [ 2025 ][ 2024 ][ 2023 ][ 2022 ][ 2021 ][ 2020 ][ 2019 ][ 2018 ][ 2017 ][ 2016 ][ 2015 ][ 2014 ][ 2013 ][ 2012 ][ 2011 ][ 2010 ][ 2009 ][ Any ]

Direction: [ Desc ][ Asc ]

• Research Paper Volume 11, Issue 8 pp 2457-2476

  Deletion of Tbk1 disrupts autophagy and reproduces behavioral and locomotor symptoms of FTD-ALS in mice

  Relevance score: 7.3599515

  Weisong Duan, Moran Guo, Le Yi, Jie Zhang, Yue Bi, Yakun Liu, Yuanyuan Li, Zhongyao Li, Yanqin Ma, Guisen Zhang, Yaling Liu, Xueqing Song, Chunyan Li

  Keywords: Tbk1, frontotemporal dementia, amyotrophic lateral sclerosis, autophagy, p62

  Published in Aging on April 30, 2019

  Show abstract
  Hide abstract

  Haploinsufficiency of the protein kinase Tbk1 has shown to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, the pathogenic mechanisms are unclear. Here we show that conditional neuronal deletion of Tbk1 in leads to cognitive and locomotor deficits in mice. Tbk1-NKO mice exhibited numerous neuropathological changes, including neurofibrillary tangles, abnormal dendrites, reduced dendritic spine density, and cortical synapse loss. The Purkinje cell layer of the cerebellum presented dendritic swelling, abnormally shaped astrocytes, and p62- and ubiquitin-positive aggregates, suggesting impaired autophagy. Inhibition of autophagic flux with bafilomycin A increased total Tkb1 levels in motor neuron-like cells in vitro, suggesting autophagy-dependent degradation of Tbk1. Although Tbk1 over-expression did not affect mutant SOD1 levels in SOD1^G93A-transfected cells, it increased the soluble/insoluble ratio and reduced the number and size of SOD1^G93A aggregates. Finally, in vivo experiments showed that Tkb1 expression was reduced in SOD1^G93A ALS transgenic mice, which showed decreased p62 protein aggregation and extended survival after ICV injection of adeno-associated viral vectors encoding Tbk1. These data shed light on the neuropathological changes that result from Tbk1 deficiency and hint at impaired autophagy as a contributing factor to the cognitive and locomotor deficits that characterize FTD-ALS in patients with Tkb1 haploinsufficiency.

  

  Conditional Tbk1-NKO mouse generation and genotyping. (A) Tbk1-NKO mice were established by crossing Tbk1^fl/fl mice with Nestin-cre mice, and genotyped by PCR. (B) Western blot expression of Tbk1 in the cortex (co), cerebellum (cb), and liver (li). (C) Tbk1 immunohistochemistry in brain cortex. Bar = 20 μm.

  
  
  

  

  Behavioral evaluation of 5-month-old Tbk1-NKO mice. (A–B) Clasping and footprint assessment. (C–D) Stretch width and stride length measurements (n = 5). (E–G) Body weight, grip power, and rotarod latency (n = 13-21). (H–I) Morris water maze’s learning and memory test. Latency to reach the platform and number of target quadrant crosses (n = 13-21). *P < 0.05, compared to WT control.

  
  
  

  

  Behavioral evaluation of 14-month-old Tbk1-NKO mice. (A–B) Latency to reach the platform and number of target quadrant crosses in the Morris water maze (n = 9-10). *P < 0.05, compared to WT control. (C–D) Swimming distances and body weight (n = 9-10). *P < 0.05, compared to WT mice.

  
  
  

  

  Dendro-synaptic alterations induced by Tkb1 deficiency. (A) NF and MAP2 immunofluorescence was used to visualize dendrites. (B) Golgi-Cox brain staining in Tbk1-NKO and WT mice (n = 3). (C) Analysis of dendritic spine density (n = 10). *P < 0.05, compared to WT mice. (D–E) Cortical synapse numbers, quantified from electron microscopy images (n = 6-7). *P < 0.05, compared to WT. Synapses are indicated by white arrows. Bar = 1 μm.

  
  
  

  

  Identification of protein aggregates. Ubiquitin, p62, and TDP-43 immunostaining of the cortex (co) and cerebellum (cb) of Tbk1-NKO and WT mice (n = 3). Bar = 20 μm. Arrows indicate protein aggregates.

  
  
  

  

  Astroglial alterations induced by Tkb1 deficiency. (A) GFAP immunostaining of the cerebellum of 5 and 14 months old mice (n = 3). Bar = 50 μm. Spheroidal astrocytes are indicated by arrows. (B) Quantification of astrocyte degeneration (n = 9); ***P < 0.001, compared to WT mice. (C) Double GFAP and p62 immunofluorescence of the cerebellum. Bar = 10 μm.

  
  
  

  

  Analysis of autophagy markers. (A–B) Western blot expression analysis of p62, LC3B, GFAP, TDP-43, and Tbk1 in the cortex (co) and cerebellum (cb) of Tbk1-NKO and WT mice (n = 3); *P < 0.05, compared to control mice.

  
  
  

  

  Assessment of neuropathological changes. (A) Bielschowsky staining of the cortex and cerebellum in mice aged 5 and 14 months (n = 3). A neurofibrillary tangle is indicated by a white arrow. Bar = 10 mm. (B–C) Quantification of dendritic densities (n = 3); *P < 0.05, compared to WT mice. (D) VGAT immunostaining of the cortex and cerebellum of mice at 5 and 14 months (n = 3). Bar = 10 mm. (E–F) Quantification of VGAT expression. Mean pixel intensity was analyzed using image J software (n = 3); *P < 0.05, compared to WT mice

  
  
  

  

  In vitro and in vivo protein expression analyses. (A–C) Western blot analysis of p-Tbk1, total Tbk1, LC3B-II, and p62 in NSC-34 cells treated with bafilomycin A, MG132, or solvent control (Con) (n = 3); *P < 0.05, compared with Con. (D) Double immunofluorescence of p-Tbk1 and p62 in the lumbar spinal cord of SOD1^G93A mice and WT littermates. Bar = 100 μm. (E–F) Western blot analysis of p62 phosphorylation status in SOD1 mutant mice and WT controls (n = 3); *P < 0.05, compared to WT mice.

  
  
  

  

  Over-expression of Tbk1 reduces the number and size of mutant SOD1 aggregates. (A–B) Western blot analysis of Tbk1, p-Tbk1, SOD1^G93A, LC3B, and p62 in NSC-34 cells transfected with mutant SOD1 (mu SOD1; i.e. SOD1^G93A) and either Tbk1 or a RFP control; representative blot (A) and summary graph of densitometric quantification data (B) (n = 3); *P < 0.05, compared to control. (C–D) Analysis of mutant SOD1 aggregates by confocal microscopy in NSC-34 cells co-transfected with mutant SOD1-GFP (mu SOD1-GFP) and Tbk1 or RFP; representative images (C) and summary graph (D) (n = 10); *P < 0.05, compared to control. (E–F) Quantification of soluble and insoluble mutant SOD1 fractions by western blot in cells over-expressing Tbk1 (n = 3); *P < 0.05, compared to control.

  
  
  

  

  Analysis of Tbk1 expression in ALS transgenic mice. (A–B) Western blot analyses of neural Tbk1 expression in SOD1^G93A transgenic mice (co: cortex, cb: cerebellum, br: brain stem, sp: spinal cord, sn: sciatic nerve; n = 3); *P < 0.05, compared to WT littermates. (C–D) Tkb1 distribution in the spinal cord evaluated by confocal microscopy. Bar = 100 μm. (E–F) Western blot analysis of phosphorylated Tbk1 in the spinal cord of SOD1^G93A mice; representative blot (E) and summary graph of densitometric analysis (F); (n = 3); *P < 0.05, compared to WT littermates. (G) Genotyping of Tbk1-NKO, SOD1G93A^+/- mice established by crossing Tbk1-NKO mice with SOD1G93A^+/- mice. (H–I) Disease symptoms onset and survival times recorded for Tbk-NKO, SOD1G93A^+/- mice; no differences were observed compared with other genotypes, i.e. Tbk1^fl/fl, SOD1G93A^+/- mice, Tbk1^fl/+, SOD1G93A^+/- mice, and Nestin-cre^+/-, Tbk1 ^fl/+, SOD1G93A^+/- mice (n = 6-7).

  
  
  

  

  Tbk1 over-expression prolongs survival of ALS mice. (A–C) Tkb1 expression and distribution in the spinal cord of SOD1^G93A mice injected (ICV) with an AAV9-Tbk1 vector (n = 3); *P < 0.05, compared to SOD1^G93A, AAV9-GFP control mice. (D) Disease onset and (E) survival rate of SOD1^G93A mice injected with AAV9-Tbk1 or AAV9-GFP control (Con) (n = 14-17); *P < 0.05, compared to control (viral titer = 1*10¹² vg/ml).