virus 2 aav2 vectors  (Addgene inc)

Journal: Acta Neuropathologica Communications

Article Title: Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration

doi: 10.1186/s40478-024-01917-6

Figure Lengend Snippet: Identification of Tppp3 by single-cell RNA sequencing. A Schematic representation of the Experimental design for single-cell RNA sequencing (scRNA-seq) conducted on E14.5 retina samples treated with GDF11 or PBS. B t t-distributed stochastic neighbor embedding (t-SNE) visualization of retinal progenitor cells, with cells color-coded based on their cluster assignments and treatment conditions. Cluster 3 represents the RGC cluster. C GDF11, RGC fate suppressor protein, leads to a reduction in the expression levels of Pou4f1 and Tppp3 , specifically within the RGC cluster (cluster 3). This highlights the potential role of Tppp3 in RGC differentiation. D Pou4f1 expression is specifically localized within cluster 3, identified as the RGC-specific cluster. E Tppp3 is also highly expressed within cluster 3. F Violin plot displaying the expression level of Tppp3 from a reanalysis of scRNA data obtained from purified RGCs. The X-axis represents the time points following optic nerve crush, while the numbers above the violin plots indicate the percentage of RGCs expressing Tppp3. 2 weeks after ONC, Tppp3 expression is reduced substantially. Tppp3 is highly expressed within the RGC clusters of G macaque and H humans

Article Snippet: Adeno-associated virus type 2 vectors driving Tppp3 overexpression- CMV > mTppp3(overexpression):P2A:EGFP (AAV2-Tppp3-OE), Tppp3 knockdown CMV > mTppp3(shRNA):P2A:EGFP (AAV2-shTppp3) or cytomegalovirus (CMV) control (AAV2-control and AAV2-shCtrl) were purchased from VectorBuilder.

Techniques: RNA Sequencing Assay, Expressing, Purification

Journal: Acta Neuropathologica Communications

Article Title: Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration

doi: 10.1186/s40478-024-01917-6

Figure Lengend Snippet: Tppp3 promotes axon regeneration and improves RGC survival. A Scheme of experimental setup for ONC and sample collection. AAV2 vectors were intravitreally injected into eyes two weeks before ONC. Two days before sample collection, CTB-555, an anterograde tracer, was intravitreally injected into eyes to label regenerating axons. Optic nerves and retinas were collected two weeks after ONC. B Representative images of optic nerve sections transduced with AAV2 vectors and labeled with CTB-555. Tppp3 overexpression significantly increases CTB-555+ axons two weeks after ONC. The crush site is marked with an asterisk. The optic nerve's proximal end (towards the eye globe) is located on the left, and the distal end (towards the brain) is on the right. Scale bar = 100 μm. C Quantification of the number of CTB-555+ axons at varying distances from the crush site after transduction with AAV2-control (n = 6 optic nerves) or AAV2-Tppp3-OE (n = 8 optic nerves). Tppp3 overexpression improves RGC regeneration at short distances from the crush site. Statistical significance was determined using an unpaired student’s t-test for each distance (** p < 0.01). Mean ± SEM is shown. D Representative images of RBPMS+ cells in flatmount retinas of (D i) negative controls without AAV transduction and ONC, or transduced with (D ii) AAV-CMV or (D iii) AAV-Tppp3-OE. Scale bar = 50 μm. E Tppp3 overexpression improves RGC survival following ONC. Quantification of the mean number of RBPMS+ RGCs in flatmount retinas after transduction with control AAV2-CMV or AAV2-Tppp3-OE, compared to the negative control without AAV2 transduction and ONC (n = 6 retinas). Statistical significance was determined using an unpaired student’s t-test (**** p < 0.0001, ** p < 0.01). Mean ± SEM is shown. F TPPP3 protein expression is significantly decreased in the optic nerve after ONC compared to sham control

Article Snippet: Adeno-associated virus type 2 vectors driving Tppp3 overexpression- CMV > mTppp3(overexpression):P2A:EGFP (AAV2-Tppp3-OE), Tppp3 knockdown CMV > mTppp3(shRNA):P2A:EGFP (AAV2-shTppp3) or cytomegalovirus (CMV) control (AAV2-control and AAV2-shCtrl) were purchased from VectorBuilder.

Techniques: Injection, Transduction, Labeling, Over Expression, Control, Negative Control, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration

doi: 10.1186/s40478-024-01917-6

Figure Lengend Snippet: TPPP3 is expressed in mouse RGCs and promotes RGC neurite outgrowth ex vivo. A Western blots show that RGC markers BRN3A, RBPMS, and THY1 are expressed selectively in the immunopurified RGC cell population, as is Tppp3. B Immunostaining of RGCs for BRN3A and TPPP3 in adult mouse retinal sections reveals that Tppp3 is expressed within the RGC layer. ~ 75% of BRN3A+ cells co-express Tppp3 (white arrows). Scale bar = 100 µm. C Immunostaining of P2 primary RGCs shows expression of anti-β-III-tubulin antibody E7. Co-labeling with anti-β-III-tubulin antibody E7 and Tppp3 confirmed that Tppp3 is expressed primarily within the soma of RGCs. Scale bar = 50 µm. D RNAscope analysis of Tppp3 in the developing mouse eye. Tppp3 expression reached its peak at E14.5 and subsequently decreased. E Representative images of primary RGCs transduced with AAV2-control or AAV2-Tppp3-OE vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 overexpression increases RGC neurite outgrowth by ~ 20% (n = 5 independent cultures). F Representative images of primary RGCs transduced with AAV2-shCtrl or AAV2-shTppp3 vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 knockdown decreases neurite outgrowth by ~ 20%. Scale bar = 100 µm. Each data point reflects an independent cell culture. Statistical significance was determined using one sample t-test (**** p < 0.0001, *** p < 0.001). Mean ± SEM is shown

Article Snippet: Adeno-associated virus type 2 vectors driving Tppp3 overexpression- CMV > mTppp3(overexpression):P2A:EGFP (AAV2-Tppp3-OE), Tppp3 knockdown CMV > mTppp3(shRNA):P2A:EGFP (AAV2-shTppp3) or cytomegalovirus (CMV) control (AAV2-control and AAV2-shCtrl) were purchased from VectorBuilder.

Techniques: Ex Vivo, Western Blot, Immunostaining, Expressing, Labeling, RNAscope, Transduction, Control, Over Expression, Knockdown, Cell Culture

Journal: Acta Neuropathologica Communications

Article Title: Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration

doi: 10.1186/s40478-024-01917-6

Figure Lengend Snippet: Tppp3 overexpression increases Bmp4 and inflammation-related genes expression. A, C Several genes related to axon regeneration and survival were upregulated after transduction of AAV2-Tppp3-OE in the whole retina two days after ONC. B Real-time qPCR data comparing changes in mRNA gene expression (relative to Gapdh housekeeping gene) showed significantly increased Bmp4 expression two days after ONC (n = 3 retinas). Statistical significance was determined using an unpaired student’s t-test (*** p < 0.001). Mean ± SEM is shown. D Identified GO terms that highlight terms related to inflammation and BMP signaling increase after Tppp3 overexpression

Article Snippet: Adeno-associated virus type 2 vectors driving Tppp3 overexpression- CMV > mTppp3(overexpression):P2A:EGFP (AAV2-Tppp3-OE), Tppp3 knockdown CMV > mTppp3(shRNA):P2A:EGFP (AAV2-shTppp3) or cytomegalovirus (CMV) control (AAV2-control and AAV2-shCtrl) were purchased from VectorBuilder.

Techniques: Over Expression, Expressing, Transduction

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: ERG Characteristics in This Study

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: Silencing of HspB8 protected RGCs and retinal function. ( A ) The AAV2-shHspB8 group had lower HspB8 protein levels (0.11 ± 0.02) compared with the control group (0.19 ± 0.02) ( P = 0.0436), as did the AAV2-shHspB8 + ONC5 group (0.19 ± 0.02) compared with the AAV2-GFP + ONC5 group (0.29 ± 0.03) ( P = 0.0089). Consistent with previous results, the ONC5 group had increased HspB8 levels (0.27 ± 0.03) compared with the control group ( P = 0.0148). No significant differences were found for the AAV2-GFP group (0.21 ± 0.02; P = 0.4383) compared with the control group or for the AAV2-GFP + ONC5 group compared with the ONC5 group ( P = 0.6461); n = 9. ( B ) Representative RGCs immunofluorescent images stained by Brn3a: ONC5 group, AAV2-shHspB8 + ONC5 group, and AAV2-GFP + ONC5 group. ( C ) The RGCs survival rate of the AAV2-shHspB8 + ONC5 group was higher (52.32% ± 2.38%) than that for the AAV2-GFP + ONC5 group (42.14% ± 1.95%) ( P = 0.0040), whereas the AAV2-GFP + ONC5 group showed no significant change compared with the ONC5 group (40.93% ± 1.32%) ( P > 0.9999; n = 6). ( D ) There were no significant differences among the visual acuities of the ONC5 group, AAV2-shHspB8 + ONC5 group, and AAV2-GFP + ONC5 group ( P = 0.3672; n = 10). ( E ) Typical ERG waves indicated that the AAV2-shHspB8 + ONC5 group had enhanced b-wave amplitudes compared with the other two groups. ( F ) The b-wave amplitudes at 3.0 and 10.0 cd·s/m² ( n = 10). ( G ) The a- and b-wave latencies demonstrated no significant changes among the three groups. The AAV2-shHspB8 + ONC5 group had higher b-wave amplitudes than those in the AAV2-GFP + ONC5 group at 3.0 and 10.0 cd·s/m² ( P = 0.0011 and P = 0.0007, respectively), whereas no difference was observed between the AAV2-GFP + ONC5 group and the ONC5 group ( P > 0.9999 and P = 0.9968, respectively). Although similar changes were found in the a-wave amplitudes, none of these differences was statistically significant. Data are shown in the ( n = 10). ( H ) Amplitudes of OPs at 3.0 cd·s/m 2 ( n = 10). Data are shown in the . All data are presented as mean ± SEM.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: Intravitreal injection of AAV2 had no impact on RGCs survival and visual function. ( A ) GFP immunofluorescent staining of retinal section in the AAV2-shHspB8 group. ( B ) Representative pictures of retinal flat-mounts stained by Brn3a in the control group, AAV2-shHspB8 group, and AAV2-GFP group. ( C ) RGCs survival rates showed no evident decrease in the AAV2-shHspB8 group (101.20% ± 2.18%) or AAV2-GFP group (100.90% ± 1.63%) compared with the control group (100.00% ± 2.30%) ( P = 0.8925 and P = 0.9367, respectively; n = 6). ( D ) Visual acuity appeared to be unaffected by intravitreal injection of AAV2-shHspB8 (0.41 ± 0.03 cyc/deg) compared with the control group (0.41 ± 0.02 cyc/deg) ( P > 0.9999; n = 10). ( E ) No differences in latencies were statistically significant (10.0 cd·s/m 2 ), nor were differences in amplitudes (3.0 and 10.0 cd·s/m², respectively) of a-waves ( P > 0.9999, P = 0.9989, and P = 0.9341, respectively) and b-waves ( P > 0.9999, P = 0.5400, and P = 0.7851, respectively) in the control group and the AAV2-shHspB8 group ( n = 10). Data are presented in the . ( F ) Neither the latency nor the amplitude of PhNRs showed significant differences between the AAV2-shHspB8 group (114.20 ± 6.51 ms, 40.64 ± 2.89 µV) and the control group (116.40 ± 5.16 ms, 36.09 ± 4.01 µV) ( P = 0.9416 and P = 0.5336, respectively; n = 7). ( G ) Example ff-ERG waves of the control group and AAV2-shHspB8 group. All data are presented as mean ± SEM.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Injection, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: The ONC enhanced autophagy level was suppressed by AAV2-shHspB8. ( A ) For the LC3II/LC3I ratios for the AAV2-shHspB8 group (0.29 ± 0.04) compared with the control group (0.37 ± 0.05), P = 0.2219; for the ONC5 group (0.52 ± 0.05) compared with the control group, P = 0.0444; for the AAV2-shHspB8 + ONC5 group (0.32 ± 0.05) compared with the AAV2-GFP + ONC5 group (0.53 ± 0.05), P = 0.0045; and for the AAV2-GFP + ONC5 group compared with the ONC5 group, P = 0.8111. For the p62 levels for the AAV2-shHspB8 group (0.98 ± 0.08) compared with the control group (0.61 ± 0.06), P = 0.0013; for the ONC5 group (0.90 ± 0.06) compared with the control group, P = 0.0170; for the AAV2-shHspB8 + ONC5 group (1.07 ± 0.07) compared with the AAV2-GFP + ONC5 (0.79 ± 0.05) group, P = 0.0260; and for the AAV2-GFP + ONC5 group compared with the ONC5 group, P = 0.7142 ( n = 9). All data are presented as mean ± SEM. ( B ) Immunofluorescent retinal sections of LC3 ( red ) and p62 ( red ); protein levels illustrated by fluorescence intensity in these five groups showed synchronized trends for the western blot results ( n = 3).

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Fluorescence, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: AAV2-shHspB8 inhibited autophagy and protected subcellular structures. N, nuclei of RGCs; white arrowheads , autolysosomes in the soma of RGCs; black # , normal mitochondria in the soma of RGCs; red # , normal mitochondria in nerve fibers; regions with blue border , unmyelinated nerve fibers and neurotubules inside; blue rectangle , normal Golgi apparatus and endoplasmic reticulum in the soma of RGCs; region with red border , apoptosis RGCs and its pyknotic nucleus; red circles , vesicular expansions of the endoplasmic reticulum; black arrowhead , autophagosome in nerve fiber. *Swollen or vacuolar mitochondria.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: AAV2-shHspB8 played a neuroprotective role by inhibiting autophagy. ( A ) Representative images of RGCs immunofluorescent staining (Brn3a). ( B ) For the ONC5-3MA group (51.53% ± 2.39%) compared with the ONC5-DMSO group (39.82% ± 2.92%), P = 0.0024; for the ONC5-Rapa group (30.71% ± 1.62%) compared with the ONC5-DMSO group, P = 0.0238; for the ONC5 group (40.93% ± 1.32%) compared with the ONC5-DMSO group, P = 0.9981; for the ONC5 group compared with the AAV2-shHspB8 + ONC5 group (52.32% ± 2.38%), P = 0.0032; for the AAV2-shHspB8 + ONC5 + Rapa group (37.95% ± 1.62%) compared with the AAV2-shHspB8 + ONC5 group, P = 0.0002 ( n = 6). ( C ) Visual acuity in the OMR test ( n = 10). ( D ) The a- and b-wave amplitudes under different flash light intensions ( n = 10). ( E ) Example ERG waves at 3.0 cd·s/m 2 . ( F ) The a-wave amplitudes at 3.0 and 10.0 cd·s/m 2 and b-wave amplitudes at 3.0 and 10.0 cd·s/m 2 : for the ONC5-3MA group compared with the ONC5-DMSO group, P = 0.0004, P = 0.4801, P = 0.0021, and P = 0.0008, respectively; for the ONC5-Rapa group compared with the ONC5-DMSO group, P = 0.0012, P = 0.0198, P = 0.0034, and P = 0.0004, respectively; for the ONC5 group compared with the ONC5-DMSO group, P = 0.0733, P = 0.3971, P = 0.5879, and P = 0.9839, respectively; for the ONC5 group compared with the AAV2-shHspB8 + ONC5 group, P = 0.1531, P = 0.2154, P < 0.0001, and P = 0.0002, respectively; and for the AAV2-shHspB8 + ONC5 + Rapa group compared with the AAV2-shHspB8 + ONC5 group, P = 0.3198, P = 0.2154, P < 0.0001, and P < 0.0001, respectively ( n = 10). Data are shown in the . ( G ) Amplitudes of OPs at 3.0 cd·s/m 2 ( n = 10). Data are shown in the .

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Staining