PMC

High-molecular-weight TDP-43 is present in different aggregation states. A and B: Sequential extraction of brain tissue showed high-molecular-weight TDP-43 (arrowheads) in detergent-soluble and detergent-insoluble fractions. C: High-molecular-weight ubiquitinated proteins were detected in LS and urea fractions (n = 6 per group).

High-molecular-weight TDP-43 interacts with VCP. A: Immunoprecipitation (IP) with a VCP antibody and immunoblot (IB) for TDP-43 uncovered an interaction between VCP and the Tg-specific TDP-43 high-molecular-weight isoform (arrowhead). B: High-molecular-weight TDP-43 is not affected by alkaline phosphatase treatment (arrowhead). C: Quantification of B (n = 4 per group). Asterisks indicate full-length TDP-43 bands. Data are given as means ± SEM.

Generation of Thy-VCP_A232E Tg mice. A: The Thy1.2-VCP_A232E transgene construct shows the location of the sequence amplified by PCR genotyping (arrows; 0.5kb) and the probe used in Southern blot analysis (2.5 kb). B: Southern blot analysis of founder lines A, B, and C. Asterisks indicate the 4- and 5.5-kb bands in Thy1.2-VCP_A232E Tg mice. Bands above 6 kb are undigested multiple copies of the transgene that were incorporated into genomic DNA. C: PCR genotyping shows amplification of 500 bp (asterisk) by the primer set shown as arrows in A. D: Immunoblot analysis of VCP in the brain. Increased steady-state levels of VCP at 98 kDa in the brain were detected in founder lines A, B, and C. Actin was used as loading control.

SHSY-5Y neuroblastoma cells present TDP-43 cytosolic buildup when transfected with mutant VCP. Cells transfected with wild-type VCP showed TDP-43 (red)–positive staining mainly in the nucleus, with little to no cytoplasmic signal (A). Cells transfected with the common R155H (B) or severe A232E (C) VCP mutation also showed TDP-43 nuclear signal with small, but evident, TDP-43 deposits in the cytoplasm (arrowheads). D–F: Green fluorescence protein signal was used to identify transfected cells. G–I: DAPI staining (blue) was used as nuclear marker. J–L: Merge images of wild-type VCP, VCP(R155H), and VCP(A232E), respectively. M: TDP-43 granule size was larger in cells transfected with mutant VCP. Scale bars: 50 μm. ^∗P < 0.05. Data are given as means ± SEM.

Thy-VCP_A232E mice display TDP-43 protein accumulation in the cytoplasm that co-localized with the stress granule marker TiA-1. A and B: Fluorescence microscopy analysis of 20-month-old mice revealed TiA-1 (red)–positive staining on wild-type (A) cortical sections but to a lesser degree than on VCP_A232E (B). D: TiA-1 co-localized with TDP-43 (green) deposits in the cytoplasm of Tg brains but not NT (C). E and F: DAPI staining (blue) was used as nuclear marker. G and H: Merge images of NT (G) and Tg (H). I: Quantification of 12- and 20-month-old sections showed significant increases in TiA-1 levels on the latter age. Data are given as means ± SEM. Scale bars: 10 μm. n = 5 to 6 per group. ^∗P < 0.05.

Thy-VCP_A232E mice show age-dependent cytosolic accumulation of polyubiquitinated proteins. A–H: VCP_A232E cortical sections from 20-month-old mice showed increased levels of polyubiquitin proteins (red) (B) and cytoplasmic TDP-43 (green) accumulation (D) compared with NT mice (A and C). E and F: DAPI staining (blue) was used as nuclear marker. G and H: Merge images of NT (G) and Tg (H). I: Quantification of 12- and 20-month-old sections uncovered increased levels of polyubiquitinated proteins on the Thy-VCP brain. J: Immunoblot analysis showed age-dependent buildup of polyubiquitinated proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. K: Polyubiquitinated proteins were accumulated in the cytosolic compartment of mutant VCP mice. Tubulin was used as loading control. L: Quantification of K. Scale bars: 10 μm. n = 5 to 6 per group. ^∗P < 0.05, ^∗∗P < 0.01. Data are given as means ± SEM.

A high-molecular-weight TDP-43 isoform accumulates in the cytoplasm of Thy-VCP_A232E mice. A: Immunoblot analysis revealed a TDP-43 band of approximately 90 kDa only on mutant VCP mice at 6 and 12 months of age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. B and C: Nuclear-cytosolic fractionation unveiled a Tg-specific TDP-43 high-molecular-weight (HMW) band in the cytosol compartment of 12- and 20-month-old animals. p84 and GAPDH were used as nuclear and cytoplasmic purity controls, respectively. D: A better resolution of the TDP-43 HMW band was obtained when samples were run in a 5% acrylamide gel. E and F: Quantification of the HMW TDP-43 band in the cytoplasmic fraction of animals aged 12 (E) and 20 (F) months (n = 6 to 7 per group). ^∗P < 0.05, ^∗∗P < 0.01. Arrowheads indicate HMW TDP-43 bands. Asterisks indicate full-length TDP-43 bands. Data are given as means ± SEM.

Overexpression of VCP_A232E causes age-dependent cognitive impairment. Six- and 12-month-old mice were trained in hippocampal- and cortical-dependent behavioral tasks. A and B: The higher-expressing VCP_A232E B line showed significant impairment in Morris water maze acquisition at 6 and 12 months of age, whereas the lower-expressing A line was not different from wild-type animals. C and D: Both 12-month-old Tg groups presented cognitive impairments when tested 24 hours later as measured by escape latency but not as measured by platform crosses. E: Both VCP_A232E-expressing lines performed poorly in the object recognition task at 12 months of age but not at 6 months of age. ^†P < 0.05 from 50% (chance level, shown as a horizontal line). F: Only the higher-expressing B line showed impairments at 6 and 12 months of age when contextual fear conditioning was assessed (n = 10 to 12 per group). ^∗P < 0.05, ^∗∗P < 0.01. Data are given as means ± SEM.