aav2 9 cag flex egfp wpre addgene  (Addgene inc)

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: AV-Mito untethering relies on AV-bound TBC1D15 GAP activity and is required for autophagic cargo clearance in tauopathy axons. ( A and B ) Representative images ( A ) and quantitative analysis ( B ) of TBC1D15 localization in normal axons. Data were expressed as the percentage of AVs or mitochondria positive for TBC1D15 and quantified from the total numbers of AVs or mitochondria ( n , indicated in parentheses) from the axons of 51 neurons ( B ). Arrows: TBC1D15-positive AVs; arrowheads: a TBC1D15-negative AV in an AV-Mito contact; asterisk: mitophagosome. ( C and D ) Representative kymographs ( C ) and quantitative analysis ( D ) of TBC1D15-tagged AVs in axons with 24-hour trehalose incubation. The relative motility of AVs was quantified from the axons of 17 neurons as indicated in parentheses ( D ). Arrows: TBC1D15-tagged motile AVs. ( E - H ) Representative kymographs ( E ) and quantitative analysis ( F - H ) of AV-Mito contacts in axons expressing TBC1D15 or TBC1D15 D397A mutant. The data were quantified and expressed as AV-Mito contact duration and its frequency, the contact number per 100 μm axonal length, the percentage of AVs in contacts, and the relative motility of AVs. Data were quantified from the total numbers of neurons ( n ) and AV-Mito contacts (c) as indicated in parentheses ( F - H ). Arrows: AV-Mito contacts. ( I and J ) Representative images ( I ) and quantitative analysis ( J ) of p62 in tauP301S axons with and without OE of TBC1D15 or TBC1D15 D397A mutant. The data were quantified and expressed as GFP-p62 puncta number per 100 μm axonal length. Data were quantified from the total numbers of neurons ( n ) as indicated in parentheses ( J ). ( K and L ) Representative blots ( K ) and quantitative analysis ( L ) of tauP301S Tg neurons transduced with and without AAV-hTBC1D15. Protein intensities were normalized to those in control tauP301S neurons without TBC1D15 OE. Data were collected from two ( B and D ), three ( F-H and J ), or five ( L ) dissections. Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B , F - H , and J ) or by two-sided Student’s t -test ( L ). Scale bars: 10 μm.

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Activity Assay, Incubation, Expressing, Mutagenesis, Transduction, Control

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: TBC1D15 OE reduces AV-Mito hyper-tethering and alleviates p62 accumulation and tau pathology in tauopathy mouse brains. ( A and B ) Representative TEM images ( A ) and quantitative analysis ( B ) of the hippocampi in 8-month-old tauP301S Tg mouse brains infected with AAV-mCherry or AAV-mCherry-hTBC1D15. The number of presynaptic AVs, the percentage of presynaptic terminals containing AVs, and the number of AVs or mitochondria in AV-Mito contacts were quantified and normalized to or compared to those in control tauP301S Tg mouse brains. Data were collected from two mice per group; the total numbers of presynaptic terminals ( n ) are indicated in parentheses ( B ). ( C and D ) Representative images ( C ) and quantitative analysis ( D ) of p62 accumulation in 10-month-old tauP301S Tg mouse hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. p62 fluorescence mean intensity in the hippocampal mossy fibers was quantified and normalized to that of AAV-mCherry-injected tauP301S Tg controls. Data were collected from three mice per group; the total numbers of imaging slice sections ( n ) are indicated in parentheses ( D ). SYP: synaptophysin. Arrows: p62 clusters at SYP-indicated presynaptic terminals. ( E and F ) Representative images ( E ) and quantitative analysis ( F ) of tau accumulation in 10-month-old non-Tg and tauP301S Tg hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. AT8 and PHF1 antibody-marked phospho-tau mean intensities in the hippocampal mossy fibers were quantified and normalized to those of AAV-mCherry-injected tauP301S Tg control mice. Data were collected from three mice per group; the total numbers of imaging slice sections ( n ) are indicated in parentheses ( F ). ( G and H ) Representative blots ( G ) and quantitative analysis ( H ) of AT8 and PHF1 antibody-marked phospho-tau and Tau5 antibody-indicated total tau in sarkosyl-extractable and sarkosyl-insoluble fractions from 10-month-old tauP301S Tg mouse brains with and without TBC1D15 OE. Protein intensities were normalized to those in AAV-mCherry-injected tauP301S Tg controls. Data were quantified from four mice per group. Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B, D and F ) or by two-sided Student’s t -test ( H ). Scale bars: 100 nm ( A ), 10 μm ( C ), and 20 μm ( E ).

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Infection, Control, Fluorescence, Injection, Imaging

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: Attenuation of neurodegeneration and memory deficits in tauopathy mice with TBC1D15 OE. (A and B ) Representative images ( A ) and quantitative analysis ( B ) of presynaptic terminal and neuron densities in 10-month-old non-Tg and tauP301S Tg hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. The mean intensity of SYP-indicated presynaptic terminals in the hippocampal mossy fibers and the number of NeuN-labeled neurons in hippocampal CA3 areas marked by rectangles (Zoomed-in images shown in the lower row of each panel in A ) were quantified and normalized to those of AAV-mCherry-injected non-Tg control mice. Data were collected from three mice per group. ( C ) Contextual fear conditioning task performed in 7-month-old non-Tg and tauP301S Tg male mice infected with AAV-mCherry or AAV-mCherry-hTBC1D15 ( n = 15-17 male mice per group). Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B) or by one-way ANOVA, followed by Sidak’s post-hoc test ( C ). Scale bars ( A) : 20 μm (upper row, SYP), 250 μm (upper row, NeuN), and 25 μm (lower row, NeuN).

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Labeling, Injection, Control, Infection