Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: CHCHD6 selectively decreases at transcription level in AD models. a Mitochondrial fractions were isolated from APP stable Neuro2a cells and subjected to Blue Native PAGE (BN-PAGE) analysis followed by western blotting (WB). MICOS complex level was detected with antibody against CHCHD6. ATPB was analyzed by SDS-PAGE followed by WB, using as a loading control. Left: representative blots from 5 independent experiments. Right: relative density of CHCHD6-immunoreactive band around 720 kDa in contrast to ATPB. b Total lysates harvested from APP stable Neuro2a cells were subjected to WB with the indicated antibodies. Left: representative blots from 3 independent experiments. Right: relative density of CHCHD6, Mitofilin and CHCHD3 in contrast to actin. c RNA was extracted from APP stable Neuro2a cells. The expression of MICOS complex components was analyzed by qPCR. Heat map analysis shows the mean of the genes analyzed. n = 6 independent experiments for CHCHD10, and n = 4 independent experiments for others. **** p < 0.0001 (Con vs. APP wt or APP swe cells). d Mitochondrial fractions were isolated from the hippocampus of WT, APP NL−G−F and APP NL−F mice at the ages of 3, 6, and 9 months, and subjected to BN-PAGE analysis. Histogram: relative density of CHCHD6-immunoreactive band around 720 kDa in contrast to ATPB. n = 4 mice/group. e The hippocampus of WT, APP NL−G−F and APP NL−F mice was harvested at the ages of 3, 6, and 9 months. Total protein levels of CHCHD6, Mitofilin and CHCHD3 were examined by WB. n = 4 mice/group. f RNA was extracted from the hippocampus of 6-month-old APP NL−G−F mice or 9-month-old APP NL−F mice and age-matched wild-type (WT) mice. The expression of MICOS complex components was analyzed by qPCR. Heat map analysis shows the mean of the genes analyzed. n = 9 mice/group for CHCHD3 of APP NL−G−F mice, and n = 5 mice/group for other groups. * p < 0.05 (WT vs. APP NL−G−F mice or APP NL−F mice). g RNA was extracted from the postmortem hippocampus of AD patients and control subjects. The expression of CHCHD6, Mitofilin and CHCHD3 was analyzed by qPCR. n = 9 individuals/group. All data are presented as mean ± SEM and were compared using one-way ANOVA with Tukey’s post hoc test in ( a – e ), and unpaired Student’s t test in ( f – g )

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Isolation, Blue Native PAGE, Western Blot, SDS Page, Expressing

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: CHCHD6 decreases in neurons of AD mice and AD patient brains. a Brain sections from WT and APP NL−G−F mice at the ages of 3, 6, and 9 months were stained with anti-CHCHD6 and anti-6E10 antibodies. The CHCHD6 and 6E10 immunodensities ( n = 3 mice/group) were quantified and shown in ( b ). c Brain sections from 6-month-old WT and 5XFAD mice were stained with anti-CHCHD6 and anti-NeuN antibodies. The CHCHD6 immunodensity in NeuN + cells ( n = 4 mice/group) was quantified and shown in ( e ). d Postmortem hippocampus sections from control subjects and AD patients were stained with anti-CHCHD6 and anti-NeuN antibodies ( n = 5 individuals/group). The intensity of CHCHD6 in NeuN + cells was quantified and shown in ( f ). DAPI was used to label nuclei. All data are presented as mean ± SEM. The data in panel b were compared by two-way ANOVA with Tukey’s post hoc test, and the data in panels e , f were compared by the unpaired Student’s t test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Staining

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: CHCHD6 and APP are interdependent to regulate each other. a . The total protein lysates of control and CHCHD6 KO HT-22 cells were subjected to immunoprecipitation (IP) with anti-APP antibody, followed by WB. Shown blots are representative of three independent experiments. b APP recombinant protein (500 ng) was incubated with either CHCHD6, Mitofilin or CHCHD3 recombinant proteins (500 ng, each). Immunoprecipitates with anti-APP antibodies was analyzed by immunoblotting with the indicated antibodies. Data are representative of 2 independent experiments. Control and CHCHD6 KO HT-22 cells were stained with anti-APP and anti-CHCHD6 antibodies ( c ) or anti-APP and anti-FACL4 antibodies ( g ), and subjected to in situ Duolink proximity ligation assay (PLA) analysis. Histogram shows the number of PLA-positive puncta (red). At least 47 cells/group (for APP and CHCHD6) and 250 cells/group (for APP and FACL4) were analyzed. The data were from 3 independent repeats. Brain sections from 6-month-old WT and APP NL−G−F mice ( n = 4 mice/group) were stained with anti-APP and anti-CHCHD6 antibodies ( d ) or anti-APP and anti-FACL4 antibodies ( h ), and subjected to PLA analysis. Histogram: the number of PLA-positive puncta (red) was quantified from at least four separate fields of each sample. e The total protein lysates of hippocampus of control subjects and AD patients were subjected to immunoprecipitation (IP) with anti-APP antibody, followed by WB. Shown blots are representative of three independent experiments. f HEK293 cells were infected with control or APP shRNA lentivirus. Total protein lysates were analyzed by WB with indicated antibodies. Histograms: relative density of CHCHD6 to actin. n = 6 independent experiments. i Control and CHCHD6 KO HT-22 cells were transfected with Flag empty vector (Flag-EV), or Flag-tagged CHCHD6 (D6-Flag) plasmids for 2 days. The total protein lysates were subjected to WB with the indicated antibodies. Histogram: the relative density of C99 and APP to actin. n = 3 independent experiments for C99 and n = 4 independent experiments for APP. j Control and CHCHD6 KO HT-22 cells were stained with anti-AICD antibody and Hoechst probe. The intensity of AICD was quantified and shown in the histogram. At least 300 cells/group were analyzed, and the data were from 4 independent experiments. k HEK293 cells were infected with control or APP shRNA lentivirus, and then transfected with the indicated plasmids for 72 h. The total protein lysates were subjected to WB. Red arrow: AICD. Histogram: the relative density of CHCHD6 to actin. n = 4 independent experiments. l HEK293 cells were transfected with CHCHD6 promoter-luc along or together with AICD, and/or Fe65 and/or Tip60 for 36 h. Cells were lysed, and the luciferase activity was measured. n = 8 for control and n = 4 for other groups. m HEK293 cells were transfected with GFP-labeled AICD, myc-Fe65 and Tip60-flag. CHIP analysis on CHCHD6 promoter was carried out with indicated antibodies. Hes1 gene ChIP is reported as a control. n = 2 independent experiments. All the data are mean ± SEM. The data in panel c , d , f – h , j were compared by the unpaired Student’s t test , and the data in panels i , k and l were compared by one-way ANOVA with Tukey’s post hoc test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Immunoprecipitation, Recombinant, Incubation, Western Blot, Staining, In Situ, Proximity Ligation Assay, Infection, shRNA, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Labeling

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: CHCHD6 deficiency induces mitochondrial impairment and cell damage. a Control and CHCHD6 KO HT-22 cells were stained with tetramethylrhodamine (TMRM) fluorescence dye. TMRM relative fluorescence density indicates the extent of mitochondrial membrane potential (MMP). Histogram: the relative fluorescence density of TMRM. At least 90 cells per group were analyzed, and the data were from 3 independent experiments. b Control and CHCHD6 KO HT-22 cells were stained with anti-Tom20 antibody (a mitochondrial marker, green) and Hoechst stain (nuclei, blue). Mitochondrial morphology was visualized using a 60X oil lens. The percentage of the cells with dot- or short bar-like fragmented mitochondria relative to the total number of cells was calculated and shown in the histogram (left). At least 50 cells per group were counted, and the data were from 3 independent experiments. Right: representative images. c Control and CHCHD6 KO HT-22 cells were treated with oligomeric Aβ 1–42 peptides (5 μM) for 48 h and subjected to MTT assay. n = 4 independent experiments. d Mitochondrial respiratory activity of control and CHCHD6 KO HT-22 cells were measured using a Seahorse XFP analyzer and a Mito stress kit. OCR oxygen consumption rate. Basal respiration rate, maximal respiration rate, and ATP production are shown. n = 3 independent experiments. e Stable APP Neuro2a cells were transfected with Flag-EV or D6-Flag plasmids for 2 days, and then subjected to 16 h serum starvation. Cell death was measurement by MTT assay. n = 5 independent experiments. f Control and CHCHD6 KO HT-22 cells were transfected with Flag-EV or D6-Flag plasmids for 2 days. Protein lysates of mitochondria fraction were analyzed by WB with the indicated antibodies. Histograms: relative density of cytochrome C to ATPB. n = 8 for EV and KO control groups; n = 5 for D6-flag overexpressed groups. g HT-22 cells were transfected with Flag-EV or D6-Flag plasmids for 24 h and then treated with oligomeric Aβ 1–42 peptides (5 μM) for 48 h. Cell death was measurement by MTT assay. n = 5 independent experiments. The data are presented as mean ± SEM. The data in panel a – d were compared by the unpaired Student’s t test, and the data in panels e – g were compared by one-way ANOVA with Tukey’s post hoc test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Staining, Fluorescence, Marker, MTT Assay, Activity Assay, Transfection

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: CHCHD6 deficiency induces neuronal cholesterol accumulation in AD models. a Control and CHCHD6 KO HT-22 cells ( n = 3) was subjected to whole transcriptome RNA-Seq analysis. Left: RNA transcripts changed in CHCHD6 KO cells. Middle: KEGG database analysis on RNA transcripts that are twofold downregulated or upregulated relative to control cells. Right: Heat map: genes involved in the “Pikuleva metabolism”. ** p < 0.01 (unpaired Student’s t test). b Control and CHCHD6 KO HT-22 cells were stained with the filipin probe. Histogram: the immunodensity of filipin + cholesterol was quantified from ten separate fields per sample. n = 3 independent experiments. c AAV5-U6-CHCHD6 shRNA-hSyn-mCherry and AAV5-U6-Scramble shRNA-hSyn-mCherry were stereotaxically injected into bilateral hippocampus of WT or APP NL−F mice at the age of 6 months. Image: AAV induced expression of mCherry in NeuN + neurons in the hippocampus of WT or APP NL−F mice 3 weeks after injection. Mice were sacrificed 6 months after injection of AAV-Scramble shRNA or AAV-CHCHD6 shRNA. d Total brain lysates were harvested from the hippocampus of 12-month-old mice at the indicated groups. CHCHD6 knockdown efficiency was assessed by WB. Histogram: the relative density of CHCHD6 to actin. n = 5 mice/group. e Brain sections from 12-month-old AAV-Scramble shRNA or AAV-CHCHD6 shRNA-injected WT or APP NL−F mice were stained with the filipin probe. The immunodensity of filipin + cholesterol was quantified and shown in f ( n = 4 mice/group). g The total cholesterol content was measured in the hippocampus from 12-month-old AAV-Scramble shRNA or AAV-CHCHD6 shRNA-injected WT or APP NL−F mice using ELISA kit ( n = 5 mice/group). h mRNAs were extracted from 12-month-old AAV-Scramble shRNA or AAV-CHCHD6 shRNA-injected WT or APP NL−F mice. Genes involved in cholesterol metabolism were analyzed by qPCR ( n = 5 mice/group). Heat map: the mean of the genes analyzed. * p < 0.05; ** p < 0.01 (AAV-Scramble shRNA-injected APP NL−F mice vs. AAV-CHCHD6 shRNA-injected APP NL−F mice. The data are presented as mean ± SEM. The data in panel d , f – h were compared by one-way ANOVA with Tukey’s post hoc test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: RNA Sequencing Assay, Staining, shRNA, Injection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: Viral vector-mediated downregulation of CHCHD6 accelerates cognitive deficits and AD pathology in APP NL−F KI mice. a The Y-maze test was performed with 12-month-old mice (WT/Scramble shRNA: n = 20 mice; n = 22 mice/group for the WT/CHCHD6 shRNA, and APP NL−F /Scramble shRNA, and APP NL−F /CHCHD6 shRNA groups). b The Barnes maze test was performed with 12-month-old mice (WT/Scramble shRNA: n = 20 mice; n = 22 mice/group for the WT/CHCHD6 shRNA, and APP NL−F /Scramble shRNA, and APP NL−F /CHCHD6 shRNA groups). c Total brain lysates were harvested from the hippocampus of 12-month-old AAV-Scramble shRNA or AAV-CHCHD6 shRNA-injected WT or APP NL−F mice. The level of C99 was analyzed by WB. Histogram: the relative density of C99 to actin ( n = 4 mice/group). d Brain sections from 12-month-old mice were stained with anti-AICD and anti-NeuN. Histogram: the intensity of AICD in NeuN + cells was quantified from four separate fields of each mouse ( n = 4 mice/group). e Brain sections from 12-month-old AAV-Scramble shRNA or AAV-CHCHD6 shRNA-injected WT or APP NL−F mice were stained with an anti-6E10 antibody. Histograms: the area covered by 6E10 + Aβ plaques and the immunodensity of 6E10 in hippocampus were quantified from four separate fields of each mouse ( n = 4 mice/group). f Brain sections of mice with the indicated groups were stained with anti-PSD95 and anti-synaptophysin antibodies. The numbers of PSD95 + and synaptophysin + puncta were quantified and shown in g ( n = 4 mice/group). h Brain sections of mice with the indicated groups were stained with anti-Iba1 and anti-GFAP antibodies. The intensity of Iba1 and GFAP was quantified and shown in ( i) ( n = 4 mice/group). The data are presented as mean ± SEM. The data in panel a , b , e , g , i were compared by one-way ANOVA with Tukey’s post hoc test, and the data in panel c and d were compared by the unpaired Student’s t test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Plasmid Preparation, shRNA, Injection, Staining

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: Compensation for the loss of CHCHD6 reduces neuropathology and cognitive deficits in APP NL−G−F KI mice. a AAV5-hSyn-CHCHD6-eGFP and AAV5-hSyn-eGFP control were stereotaxically injected into bilateral hippocampus of 3-month-old WT or APP NL−G−F mice. Image: AAV induced expression of eGFP in NeuN + neurons in mouse hippocampus 3 weeks after injection. Mice were killed 6 months after AAV injection. b Total brain lysates were harvested from the hippocampus of 9-month-old mice. WB was performed with the indicated antibodies. Histogram: the relative density of endogenous CHCHD6 to actin ( n = 5 mice/group). c The Y-maze test was performed with 9-month-old mice (WT/EV and APP NL−G−F /CHCHD6 groups: n = 25 mice; n = 26 mice for WT/CHCHD6; and n = 24 mice for APP NL−G−F /EV). d The Barnes maze test was carried out with 9-month-old mice (WT/EV and APP NL−G−F /CHCHD6 groups: n = 25 mice; n = 26 mice for WT/CHCHD6; and n = 24 mice for APP NL−G−F /EV). e mRNAs were extracted from 9-month-old mice at the indicated groups. Genes involved in cholesterol metabolism were analyzed by qPCR ( n = 5 mice/group). Heat map: the mean of the genes analyzed. * p < 0.05; *** p < 0.001; **** p < 0.0001; (AAV-EV-injected APP NL−G−F mice vs. AAV-CHCHD6-injected APP NL−G−F mice). f Brain sections from 9-month-old mice at the indicated groups were stained with the filipin probe. The immunodensity of filipin + cholesterol was quantified and shown in ( g ) ( n = 4 mice/group). h The total cholesterol content was measured in the hippocampus from 9-month-old mice using ELISA kit ( n = 6 mice/group). i Total brain lysates were harvested from the hippocampus of 9-month-old mice. The level of C99 was analyzed by WB. Histogram: the density of C99 relative to actin ( n = 4 mice/group). j Brain sections from 9-month-old AAV-CHCHD6 or AAV-EV-injected APP NL−G−F mice were stained with anti-AICD and anti-NeuN. The intensity of AICD in NeuN + cells was quantified from four separate fields of each mouse ( n = 4 mice/group). The data in panel b – e , g , h were compared by one-way ANOVA with Tukey’s post hoc test, and the data in panel i , j were compared by the unpaired Student’s t test

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Injection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica

Article Title: A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease

doi: 10.1007/s00401-022-02499-0

Figure Lengend Snippet: Compensation for the loss of CHCHD6 reduces amyloid accumulation, synaptic loss and gliosis in APP NL−G−F KI mice. a Brain sections from 9-month-old AAV-CHCHD6 or AAV-EV-injected APP NL−G−F mice were stained with anti-6E10 antibodies. Histograms: the area covered by 6E10 + Aβ plaques in hippocampus were quantified from four separate fields of each mouse ( n = 4 mice/group). b Mouse brain sections were stained with anti-PSD95 and anti-synaptophysin antibodies. Histograms: the numbers of PSD95 + and synaptophysin + puncta were quantified ( n = 4 mice/group). c Mouse brain sections were stained with anti-Iba1 antibody. Histograms: the intensity of Iba1 was quantified ( n = 5 mice/group). The data are presented as mean ± SEM. The data in panel a were compared by the unpaired Student’s t test, and the data in panel b and c were compared by one-way ANOVA with Tukey’s post hoc test. d A summarized scheme

Article Snippet: To silence CHCHD6 in HT-22 cells, a control and CHCHD6 synthetic guide RNA (sgRNA) CRISPR lenti-vector set (K3156005) was purchased from Applied Biological Materials (Richmond, BC, Canada).

Techniques: Injection, Staining

Journal: Journal of Nanobiotechnology

Article Title: Short cell-penetration peptide conjugated bioreducible polymer enhances gene editing of CRISPR system

doi: 10.1186/s12951-024-02554-w

Figure Lengend Snippet: TSP scp promotes gene activation activity of CRISPR-dCas system. ( A ) Activation of mNG expression by mCherry-dCas12f-VPR/sgRNA targeting mNG promoter in the HEK293T reporter cells after TSP scp /pDNA, TSP/pDNA, PEI/pDNA and Lipo/pDNA transfection. In the non-targeted (NT) sgRNA control, cells transfected with a non-targeted sgRNA. Fluorescence imaging and flow cytometry analysis were performed 48 h post transfection. Red, mCherry-dCas12f-VPR. Green, mNG. VPR (VP64-p65-Rta), a transcriptional activator. Scale bar, 250 μm. ( B ) Schematic analysis of endogenous gene activation by CRISPR-dCas12a delivered by TSP scp and other transfection reagents. ( C-D ) qPCR analysis of targeted genes including HBG, IL1RN and TTN, in TSP scp /pDNA, TSP/pDNA, PEI/pDNA and Lipo/pDNA transfected cells. RNA extraction was performed 48 h post transfection. n = 3 per group. Data represents as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. P -values are from ANNOVA analysis and Sidak’s multiple comparisons test

Article Snippet: Plasmids expressing single guide RNA (sgRNA) were constructed by ligating the corresponding annealed oligos to the basic plasmid under the human U6 promoter (GENEWIZ, China).

Techniques: Activation Assay, Activity Assay, CRISPR, Expressing, Transfection, Fluorescence, Imaging, Flow Cytometry, RNA Extraction