Fig 1: DNA damage from NSCs to adult neurons of VCPT262A-KI mice.(A) Time-lapse imaging of VCP-EGFP assembly in linear irradiated areas containing double-strand breaks. After transfection with VCP-EGFP or VCPT262A-EGFP, the nuclei of U2OS cells were micro-irradiated, and assemblies of VCP-EGFP and VCPT262A-EGFP were observed. Right graph shows quantitative analysis of the recovery of VCP-EGFP signals in the linear bleached areas. At each time-point after micro-irradiation, EGFP signal intensities were quantified and normalized to the area signal before micro-irradiation (white box). Means and s.e.m. are indicated. Asterisks indicate statistical differences (P < 0.01, t test). (B) Immunohistochemistry with anti-?H2AX or anti-53BP1 antibodies in cerebral cortex (VZ/SVZ) at E13 and E15. Right graph shows percentage of VZ/SVZ cells with ?H2AX or 53BP1 foci. (C) Western blot analyses of ?H2AX and 53BP1 in cortex tissue at E15. (D) Western blot analyses of ?H2AX and 53BP1 in NSC spheres prepared from embryonic brains of heterozygous VCPT262A-KI and C57BL/6J mice at E15. Right graphs show quantitative analysis of band intensities. (E) Immunohistochemistry and Western blot of double-strand break markers ?H2AX and 53BP1 reveal elevated DNA damage in adult cerebral neurons of VCPT262A-KI mice at 3 and 6 mo. (F) Immunohistochemistry of phosphorylated TDP43 in cerebral cortex neurons of VCPT262A-KI mice at 6 and 12 mo. (G) Immunohistochemistry of ?H2AX and phosphorylated TDP43 in postmortem brains of human FTLD patients. ?H2AX was barely detectable in control brains (non-neurological disease patient, N = 6). Representative images of double-positive neurons and ?H2AX-positive/pTDP43-negative neurons from FTLD patients (N = 5) are shown. Tables show classification of staining patterns in human cortical neurons in frontal lobe of three controls and three FTLD patients. Staining patterns of ?H2AX and pTDP43 in MAP2-positive neurons were positively related (Fisher’s exact test, P = 2.2 × 10-16). (H) Western blot analysis of ?H2AX in postmortem brains of human FTLD patients and controls. Box plots show medians, quartiles, and whiskers, which represent data outside the 25th–75th percentile range.Source data are available for this figure.
Fig 2: Morphological characterization of VCPT262A-KI mice.(A) Immunohistochemistry with anti-TDP43 antibody revealed that cytoplasmic TDP43 levels were elevated in neurons of VCPT262A-KI mice. Panels show representative images in layers III–IV of frontal association cortex (FrA) at 6 mo of age. Red arrow in upper panels indicates a neuron magnified in lower panels, and black arrow indicates cytoplasmic aggregates. (B) Immunohistochemistry with anti-ubiquitin antibody revealed that the similar neurons were positively stained by ubiquitin in VCPT262A-KI mice. (C) p62 inclusion bodies were detected at a low frequency in VCPT262A-KI mice. (D) Fused in sarcoma inclusion bodies were detected at a low frequency in VCPT262A-KI mice. (E) Summary of pathological findings in VCPT262A-KI mice. (F) TDP43-positive or ubiquitin-positive inclusion bodies were not found in embryonic cerebral cortex, especially in VZ-containing NSCs.
Fig 3: Non-phosphorylated MCM3 rescues phenotypes of VCPT262A-KI mice.(A) Experimental protocol for AAV injection into mouse embryos and subsequent examinations. AAV-pNestin-VCP-FLAG, AAV-pNestin-MCM3T719A-FLAG, and AAV-pNestin-FLAG were injected into pregnant mice at E10 via the tail vein (1.0 × 1012 vg in 100 µl). Newborn mice were reared by their mothers until weaning, and then their genotypes were examined. At the ages of 1 or 6 mo, brains were dissected, weighed, and subjected to immunohistochemistry. At the age of 6 mo, behavioral tests (Y-maze test and Morris water maze test) were performed. (B) Total brain weight of VCPT262A-KI mice (male) treated at E10, from 1 to 6 mo of age. (C) Coronal sections of brains of VCPT262A-KI mice at 6 mo. At 1 mo, AAV-pNestin-VCP-FLAG and AAV-pNestin-MCM3T719A-FLAG, but not AAV-pNestin-FLAG, rescued brain size. (D) Abnormal cortical layer structure was normalized by AAV-pNestin-VCP-FLAG and AAV-pNestin-MCM3T719A-FLAG. Cux1 (layers II–III), Foxp1 (layers III–V) and Tbr1 (Layers V–VI) were used as layer markers. (E) Immunohistochemistry of DNA damage markers in VCPT262A-KI mice treated at E10, at 6 mo of age. (F) Western blotting for DNA damage markers in VCPT262A-KI mice treated at E10 at 6 mo of age. (G) Morris water maze test and Y-maze test showed recovery of spatial memory impairment at 6 mo of age in VCPT262A-KI mice injected with AAV-pNestin-VCP-FLAG or AAV-pNestin-MCM3T719A-FLAG at E10. Graphs show latency to platform at day 5 of training in the Morris water maze test and alteration rate in the Y-maze test. #P < 0.05, ##P < 0.01 (Dunnett’s test). (H) Protocol for gene therapy in VCPT262A-KI mice at 5 mo (adulthood). (I) Immunohistochemistry for DNA damage markers was performed in 6-mo-old VCPT262A-KI mice that received gene therapy at 5 mo. AAV-pCMV-VCP-FLAG, but not AAV-pCMV-MCM3T719A-FLAG, decreased DNA damage in cortical neurons. (J) Morris water maze test and Y-maze test showed recovery of spatial memory impairment at 6 mo of age in VCPT262A-KI mice injected with AAV-pCMV-VCP-FLAG or AAV-pCMV-MCM3T719A-FLAG at E10 at 5 mo of age. Graphs show latency to platform at day 5 of training in the Morris water maze test and alternation rate in the Y-maze test. #P < 0.05, ##P < 0.01 (Dunnett’s test). (K) TDP43 aggregation in cortical neurons at 6 mo of VCPT262A-KI mice that were treated with AAV-pNestin-VCP-FLAG and AAV-pNestin-MCM3T719A-FLAG at E10 or with AAV-pCMV-VCP-FLAG and AAV-pNestin-MCM3T719A-FLAG at 5 mo. The number of cells with TDP43 aggregation decreased significantly in mice that received gene therapy at E10. Box plots show median, quartiles, and whiskers, which represent data outside the 25th–75th percentile range.Source data are available for this figure.
Fig 4: Generality of neuronal TRIAD necrosis across other FTLD mouse models and human FTLD patients.(A) Immunohistochemistry of YAP, pSer46-MARCKS, damaged DNA (nick-end-labeling) in cerebral cortex of three FTLD mouse models other than VCPT262A-KI mice and their background (C57BL/6J) at 1 mo of age. (B) Chronological changes in the frequency of TRIAD necrosis in cerebral cortex of three FTLD mouse models other than VCPT262A-KI mice. Necrotic neurons were counted in randomly selected visual fields (n = 10, N = 3, 143 × 143 µm). Representative images of necrotic neurons associated with pSer46-MARCKS are shown in the lower panels. (C) Western blot reveals elevated levels of pSer46-MARCKS in cerebral cortex of PGRNR504X-KI mice during aging. (D) Nissl staining revealed chromatolysis of residual neurons in occipital lobes of human FTLD patients. (E) KDEL, an ER marker, reveals remarkable ER expansion in neurons of non-familial sporadic FTLD patients (left upper panels). Such neurons were accompanied by pSer46-MARCKS staining, and DAPI staining became weak (right upper panels). YAP signals were reduced in neurons with ER expansion and immunoreactivity for pSer46-MARCKS (left lower panel). (F) Electron microscopy images confirmed ER expansion of necrotic neurons in cerebral cortex of FTLD-KI mice (white and yellow arrows). High-magnification images of vacuoles (yellow arrow) are shown. (G) Co-staining of pSer409/410-TDP43 and pSer46-MARCKS of a neuron. (H) Western blots of TDP43 protein after in vitro phosphorylation by PKC? or CK1. Antibody against TDP43 detected both phosphorylated (white arrow) and non-phosphorylated TDP43 (black arrow), whereas specific antibody against pSer403/404 or pSer409/410 detected only TDP43 phosphorylated by PKC? or CK1. (I) Western blots of four FTLD mouse models revealed changes in TDP43 phosphorylation and total YAP level during aging. YAP level was reduced in FTLD model mice relative to non-Tg sibling mice, and also decreased during aging. (J) Upper panels: Rescue of DNA damage by in utero treatment with AAV expressing non-phosphorylated mutant MCM3 (AAV-pCMV-MCM3T719A-FLAG) in four FTLD models. Middle panels: Rescue of TRIAD necrosis by in utero treatment with AAV expressing non-phosphorylated mutant MCM3 (AAV-pCMV-MCM3T719A-FLAG) in four FTLD models. Lower panels: Rescue of TDP43 aggregation by in utero treatment with AAV expressing non-phosphorylated mutant MCM3 (AAV-pCMV-MCM3T719A-FLAG) in four FTLD models. (K) Rescue of cognitive impairment by in utero treatment with AAV expressing non-phosphorylated mutant MCM3 (AAV-pCMV-MCM3T719A-FLAG) in four FTLD models. Upper and lower panels show the results of the Y-maze test and Morris water maze test, respectively. Box plots show medians, quartiles, and whiskers, which represent data outside the 25th–75th percentile range.Source data are available for this figure.
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