AAV ablates neurogenesis in the adult murine hippocampus
- Neurosciences Graduate Program, University of California, San Diego, United States
- Laboratory of Genetics, Salk Institute for Biological Studies, United States
- Department of Neurosciences, University of California, San Diego, United States
- Department of Biology, University of California, San Diego, United States
- Department of Physics, University of California, San Diego, United States
- Laboratory of Experimental Epileptology, Department of Physiology, Second Faculty of Medicine, Charles University, United Kingdom
- Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine, Albert Einstein College of Medicine, United States
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 1 supplement
rAAV eliminates abDGCs in a dose-dependent manner.
(A) Experimental design of rAAV injection into DG following indelible labeling of adult-born DGCs with BrdU. (B, left) Representative images showing Prox1 and BrdU used for quantification. (B, …
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Figure 1—source data 1
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Figure 1—figure supplement 1
rAAV-dependent toxicity.
(A) The amount of reduction in DCX expression induced by rAAV is variable at 2 weeks post-injection but is present despite differences in viral core (Salk, Upenn), purification method (CsCl, …
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Figure 1—figure supplement 1—source data 1
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Figure 2 with 2 supplements
Developmental stage determines susceptibility to rAAV-induced cell loss.
(A) Experimental design to assess the effect of rAAV post-injection interval on the survival of different NPC types. Following labeling with BrdU, mice are injected unilaterally with 1 µL of 3 e12 …
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Figure 2—source data 1
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- https://cdn.elifesciences.org/articles/59291/elife-59291-fig2-data1-v2.zip
Figure 2—figure supplement 1
Cell-type-specific cell loss.
(A–F) Co-localization of GFP and neurogenesis markers 1 week after ipsilateral injection of AAV1-CAG-GFP into the DG without BrdU. Animals also received contralateral saline injection. (A, B) …
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Figure 2—figure supplement 1—source data 1
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Figure 2—figure supplement 2
Inflammation and cell loss.
(A) Representative images of increased microglia activation 4 weeks after rAAV injection. (B) Extending experiments from Figure 2A–E, Iba1 intensity is not significantly changed in subgranular zone …
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Figure 2—figure supplement 2—source data 1
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Figure 3 with 1 supplement
rAAV induces toxicity in NPCs in vitro.
(A) Dose-dependent inhibition of NPC proliferation by rAAV; initial multiplicity of infectivity (MOI) of 1 e7 viral particles/cell arrests mNPC proliferation by 24 hr. MOI of 1 E6 results in slower …
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Figure 3—source data 1
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Figure 3—figure supplement 1
FLOW Cytometry gating strategy for analysis of AAV ITR-induced cell death during cell cycle.
(A) Representative gating strategy for selection of single and dividing cells. Example for PBS sample shown in B. (B) Representative two-parameter density plots used to determine population …
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Figure 3—figure supplement 1—source data 1
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Figure 4 with 1 supplement
AAV retro serotype permits studies of DGC activity in vivo without ablating adult neurogenesis.
(A) Representative images showing cFos and Dapi used for quantification. (B) Mature DGCs are hyperactive following rAAV-induced cell loss in a dose-dependent manner for injected titers between 3e12 …
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Figure 4—source data 1
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- https://cdn.elifesciences.org/articles/59291/elife-59291-fig4-data1-v2.zip
Figure 4—figure supplement 1
Calcium imaging following 2 week knockdown of adult neurogenesis.
(A) Additional analysis of experiments described in Figure 4A,B demonstrate that cFOS activation in mature DGCs is significantly correlated with knockdown efficiency of adult neurogenesis (n = 23 …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
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- https://cdn.elifesciences.org/articles/59291/elife-59291-fig4-figsupp1-data1-v2.zip
Tables
Table 1
DG Injection coordinates.
Injection coordinates as measured from bregma adjusted for measured distance between lambda and bregma (Λ-B): anterior–posterior (A/P), medial–lateral (M/L); and dorsoventral depth from dura (D/V).
| Λ-B [mm] | A/P [mm] | M/L [mm] | D/V [mm] |
|---|---|---|---|
| 3.0 | −1.5 | ±1.5 | −1.8 |
| 3.2 | −1.6 | ±1.55 | −1.8 |
| 3.4 | −1.7 | ±1.6 | −1.9 |
| 3.6 | −1.8 | ±1.65 | −1.9 |
| 3.8 | −1.9 | ±1.7 | −1.95 |
| 4.0 | −2.0 | ±1.75 | −2.0 |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | #000664 | |
| Strain, strain background (Mus musculus) | STING-KO (B6(Cg)-Sting1tm1.2Camb/J ) | The Jackson Laboratory Jin et al., 2011 | #025805 | |
| Chemical compound, drug | BrdU (5-Bromo-2’-deoxyuridine) | Millipore Sigma | B5002 | 50 mg/kg/day i.p. for 3 days |
| Other | Nanoject III | Drummond Science | 3-000-207 | |
| Strain, strain background (AAV) | AAV1-CAG-GFP | Addgene | 37825 | |
| Strain, strain background (AAV) | AAV1-CAG::flex-eGFP-WPRE-bGH | Addgene and University of Pennsylvania | 51502 | |
| Strain, strain background (AAV) | AAVretro-CaMKIIa::NES-jRGECO1a-WPRE-SV40 | Gage, Salk Viral Core | ||
| Strain, strain background (AAV) | AAV8-CaMKIIa::NES-jRGECO1a-WPRE-SV40 | Gage, Salk Viral Core | ||
| Strain, strain background (AAV) | AAV1-Syn::NES-jRGECO1a-WPRE-SV40 | Addgene and University of Pennsylvania | 100854 | |
| Strain, strain background (AAV) | AAV8-CaMKIIa::mCherry-WPRE-bGH | Addgene and Salk Viral Core | 114469 | |
| Strain, strain background (AAV) | AAV8 capsid | University of North Carolina Viral Vector Core pXR8, Salk Viral Core | ||
| Other | 3 mm glass coverslip | Warner Instruments | CS-3R | #1 thickness |
| Other | Optibond All-In-One | Kerr Dental | 33381 | |
| Other | 2-Photon Laser Scanning Microscope Movable Objective Microscope (MOM) | Sutter Instruments | ||
| Other | 1070 nm femtosecond-pulsed laser | Coherent | Fidelity-2 | |
| Other | Nikon 16X water immersion objective | 0.8 NA, Nikon | CFI75 LWD 16X W | |
| Software, algorithm | Matlab | MathWorks | ||
| Software, algorithm | FlowJo | BD Biosciences | ||
| Software, algorithm | Prism 9.0 | GraphPad | ||
| Antibody | Anti-BrdU (rat monoclonal) | Accurate. Novus, Abcam | OBT0030, Accurate; NB500-169, Novus; AB6326, Abcam | IHC (1:250) |
| Antibody | Anti-cleaved-CASPASE3 (rabbit polyclonal) | Cell Signaling | 9661 | IHC (1:200) |
| Antibody | Anti-CFOS (goat polyclonal) | Santa Cruz | sc-52-G | IHC (1:500) |
| Antibody | Anti-CFOS (rabbit polyclonal) | Synaptic Systems | 226003 | IHC (1:500) |
| Antibody | Anti-DCX (goat polyclonal) | Santa Cruz | sc-8066 | IHC (1:50–500) |
| Antibody | Anti-DCX (guinea pig polyclonal) | Millipore | AB2253 | IHC (1:500) |
| Antibody | Anti-GFAP (chicken polyclonal) | Millipore | AB5541 | Ihc (1:1000) |
| Antibody | Anti-GFP (chicken polyclonal) | Aves Labs | GFP-1020 | IHC (1:1000) |
| Antibody | Anti-PROX1 (rabbit polyclonal) | Abcam | ab101851 | IHC (1:500) |
| Antibody | Anti-SOX2 (rabbit polyclonal) | Cell Signaling | 2748 | IHC (1:250) |
| Antibody | Anti-TBR2 (rabbit monoclonal) | Abcam | ab183991 | IHC (1:500–1000) |
| Antibody | Anti-SOX2 (rat monoclonal) | Invitrogen | 14981182 | IHC (1:1000) |
| Antibody | Anti-chicken-AlexaFluor488 (donkey polyclonal) | Jackson Immuno Research Laboratories | 703-545-155 | IHC (1:250) |
| Antibody | Anti-rat-AlexaFluor647 (donkey polyclonal) | Jackson Immuno Research Laboratories | 712-605-153 | IHC (1:250) |
| Antibody | Anti-rabbit-Cy5 (donkey polyclonal) | Jackson Immuno Research Laboratories | 711-175-152 | IHC (1:250) |
| Antibody | Anti-rabbit-Cy3 (donkey polyclonal) | Jackson Immuno Research Laboratories | 711-165-152 | Ihc (1:250) |
| Antibody | Anti-rabbit-AlexaFluor488 (donkey polyclonal) | Jackson Immuno Research Laboratories | 711-545-152 | IHC (1:250) |
| Antibody | Anti-guinea pig-AlexaFluor488 (donkey polyclonal) | Jackson Immuno Research Laboratories | 706-545-148 | IHC (1:250) |
| Antibody | Anti-guinea pig-Cy3 (donkey polyclonal) | Jackson Immuno Research Laboratories | 706-165-148 | IHC (1:250) |
| Antibody | Anti-guinea pig-AlexaFluor647 (donkey polyclonal) | Jackson Immuno Research Laboratories | 706-605-148 | IHC (1:250) |
| Antibody | Anti-goat-Cy5 (donkey polyclonal) | Jackson Immuno Research Laboratories | 705-175-147 | IHC (1:250) |
| Antibody | anti-goat-Cy3 (donkey polyclonal) | Jackson Immuno Research Laboratories | 705-165-147 | IHC (1:250) |
| Antibody | Anti-goat-AlexaFlour488 (donkey polyclonal) | Jackson Immuno Research Laboratories | 705-545-147 | IHC (1:250) |
| Other | Zeiss laser scanning confocal microscope | Zeiss | LSM 710, LSM 780, or Airyscan 880 | 20x and 63x objective |
| Other | Slide scanning microscope | Olympus | VS-120 | 10× objective |
| Other | Libra 120kV PLUS EF/TEM | Carl Zeiss | 2kx2k CCD camera, 20,000x magnification | |
| Commercial assay or kit | Amaxa Mouse NSC Nucleofector Kit | Lonza | VPG-1004 | |
| Sequenced-based reagent | ITR | Integrated DNA Technologies | 20nmole Ultramer DNA Oligo | 5’-Biotin- AGGAACCCC TAGTGATGGAGTTGG CCACTCCCTCTCTGCG CGCTCGCTCGCTCAC TGAGGCCGGGCGACC AAAGGTCGCCCGACG CCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCG AGCGAGCGCGCAGAGA GGGAGTGGCCAA-3’ |
| Sequenced-based reagent | ITR Scramble | Integrated DNA Technologies | 20nmole Ultramer DNA Oligo | 5’-Biotin-CCACATACCGT \CTAACGTACGGATTCC GATGCCCAGATATATAG TAGATGTCTTATTTGTG GCGGAATAGCGCCAGAG CGTGTAGGCCAACCTTA GTTCTCCATGGAAGGCA TCTACCGAACTCGGTTGC GCGGCCAAATTGGAT-3’ |
| Chemical compound, drug | Vybrant DyeCycle Green Stain | Thermo Fisher | V35004 | 1:2000 |
| Chemical compound, drug | Zombie UV Fixable Viability Kit | BioLegend | 423107 | 1:1000 |
| Other | CountBright Absolute Counting Beads | Thermo Fisher | C36950 | ~5000 beads/sample |
| Other | LSRFortessa X-20 | BD Biosciences and UCSD Human Embryonic Stem Cell Core |
Additional files
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Supplementary file 1
Tukey’s test for group comparisons of AAV ITR and SCR control.
Post hoc comparisons of the electroporation groups presented in Figure 3E,F following two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant.
- https://cdn.elifesciences.org/articles/59291/elife-59291-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59291/elife-59291-transrepform-v2.docx
