aav2 2 vector  (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: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Deletion of Pten and Socs3 increases axotomy-induced axonal regeneration in primary RGCs. (A) Primary retinal cultures are obtained from retinae of Pten fl/fl ;Socs3 fl/fl mice at P2–3 and seeded in microfluidic devices. The RGCs are labeled and conditionally deleted for Pten and Socs3 via AAV2/2-mediated transduction at 1DIV. Axonal injury is performed between 12DIV and 14DIV and the outcome of regeneration is assessed 1 day later (1 dpi, DIV12–14+1). (B) Representative images of uninjured axons (naive), injured axons immediately after axotomy (0 dpi), and regrowing axons (1 dpi) of WT and cdKO RGCs. hSyn-YFP is encoded by an AAV2/2-hSyn-ATeam YEMK -WPRE-hGHp vector (see Materials and methods). Scale bar 100 µm. (C) Representative images of degenerating axons (red), regenerating axons (green), and static axons (orange). The latter survive injury but do not grow past the cut site at 1 dpi and until 3 dpi. Scale bar 50 µm. (D) Quantification of the axons regrowing in the axonal compartment shows that deletion of Pten and Socs3 induces an increase in the percentage of regenerating axons at 1 dpi. (E) Quantification of the axonal length shows that codeletion of Pten and Socs3 induces a significant increase in the average length of regrowing axons at 1 dpi. The median length is reported above both groups. (F) Cumulative distribution of axonal lengths shows that Pten and Socs3 co-deletion increases the length of regrowth at 1 dpi. Data from six independent experiments, presented as mean ± SD (D) or median ± 25–75th confidence interval (E) and bootstrap 95% confidence interval versus WT. Student’s t test (D), Kolmogorov–Smirnov test (E), and Mann–Whitney U test (F). P values are reported within the graphs.

Article Snippet: For mitochondrial imaging, cells were transduced with a Cre-dependent AAV2/2-CAG-FLeX-mitoGFP-t2A-mCherry-WPRE vector (Charles River, 2.7 × 10 10 GC/ml final titer in medium).

Techniques: Labeling, Transduction, Plasmid Preparation, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: AAV-mediated conditional knockout of Pten and Socs3 leads to specific deletion and upregulation of regeneration-associated pathways at 14DIV. (A) Immunolabelling for DAPI, PTEN, and Tuj1 shows specific loss of PTEN within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector (cdKO) as compared to WT cells, indicating specific recombination and gene knockout. Scale bar 50 µm. (B) Quantification of PTEN signal in the somata of hSyn-YFP + RGCs shows that on average 98.7% of the RGCs are positive for PTEN at 14DIV in the WT condition as compared to only 2% in the cdKO culture, indicating that an efficient knockout is achieved. (C) Immunolabeling for DAPI, pS6, and Tuj1 reveals increased phosphorylation of S6 within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector as compared with WT cells, indicating mTOR activation. Scale bar 50 µm. (D) Quantification of pS6 signal in the somata of hSyn-YFP + RGCs at 14DIV shows a ∼30% increase in the number of pS6 + RGCs upon deletion of Pten (P) and Socs3 (S). (E) Immunolabeling for DAPI, pSTAT3, and Tuj1 reveals increased phosphorylation of STAT3 within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector as compared to WT cells, indicating JAK/STAT activation. Scale bar 50 µm. (F) Quantification of pSTAT3 signal in the somata of hSyn-YFP + RGCs at 14DIV shows a ∼90% increase in the number of pSTAT3 + RGCs upon deletion of Pten and Socs3 . In all experiments hSyn-YFP was encoded by AAV2/2-hSyn1-ATeam YEMK -WPRE-hGHp ; see Materials and methods. Data from four (A and B) or two (C–F) independent experiments, presented as mean ± SD (B, D, and F) and bootstrap 95% confidence interval versus WT (B, D, and F). Student’s t test (B, D, and F). P values are reported within the graphs.

Article Snippet: For mitochondrial imaging, cells were transduced with a Cre-dependent AAV2/2-CAG-FLeX-mitoGFP-t2A-mCherry-WPRE vector (Charles River, 2.7 × 10 10 GC/ml final titer in medium).

Techniques: Knock-Out, Transduction, Plasmid Preparation, Gene Knockout, Immunolabeling, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Deletion of Pten and Socs3 increases axonal glycolytic energy after injury. (A) Comparison of non-normalized axonal ATP measurements does not identify any significant difference in the intra-axonal concentration of ATP in naive axons between WT and cdKO neurons. (B) Comparison of non-normalized axonal lactate measurements does not identify any significant difference in the intra-axonal concentration of lactate in naive axons between WT and cdKO neurons. (C) Representative images of axons, after PFKP (Phosphofructokinase, Platelet) immunolabeling (magenta), assigned to each different scoring category. Axons with no appreciable labeling are classified as low. Faint labeling is scored as mid, while bright labeling and the presence of foci are scored as high. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp; see Materials and methods. Scale bar 10 µm ( D) Semiquantitative analysis via randomized blind scoring of PFKP immunolabeled axons by two independent evaluators reveals increased labeling in regenerating cdKO axons as compared to WT ones. n > 20 regenerating axons for both genotypes. (E) Representative images for each scoring category of PKM (Pyruvate Kinase M1/2) immunolabeled axons (magenta). Axons with faint signal are classified as low. Labeling with sparse foci is classified as mid, while bright labeling and the presence of abundant foci is scored as high. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp. Scale bar 10 µm. (F) Semiquantitative analysis via randomized blind scoring of PKM immunolabeled axons by two independent evaluators shows increased labeling in regenerating cdKO axons as compared with WT ones. n > 20 regenerating axons for both genotypes. (G) Representative images of axons, after LDHA (Lactate Dehydrogenase A) immunolabeling (magenta), were assigned to each different scoring category. Axons are classified from low to high based on brightness and uniformity of the labeling along the distal end of the axon. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp. Scale bar 10 µm. (H) Semiquantitative analysis via randomized blind scoring by two independent evaluators reveals increased LDHA labeling in regenerating cdKO axons as compared with WT ones. n > 20 regenerating axons for both genotypes. (I) Relative percentage representation of mitochondrial ATP (oligomycin sensitive, green, top) and glycolytic ATP (oligomycin and 2-deoxyglucose sensitive, red, bottom) at 1 dpi. Both static and regenerating cdKO axons show a higher relative percentage of glycolytic ATP as compared to WT axons. The percentage value was obtained by subtracting the baseline measurement in the presence of both 10 µM oligomycin and 50 mM 2-deoxyglucose from the measurements in normal medium and oligomycin-only. Then, the oligomycin-only measurement was expressed as a percentage of the measurement in normal medium. Data from five (A), four (B and I) independent experiments, presented as median ± 25–75th confidence interval (A and B), mean ± SD (D–H), or mean ± SEM (I) and bootstrap 95% confidence interval versus WT (A and B). Mann–Whitney U test (A and B). P values are reported within the graphs.

Article Snippet: For mitochondrial imaging, cells were transduced with a Cre-dependent AAV2/2-CAG-FLeX-mitoGFP-t2A-mCherry-WPRE vector (Charles River, 2.7 × 10 10 GC/ml final titer in medium).

Techniques: Comparison, Concentration Assay, Immunolabeling, Labeling, Imaging, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Regenerating cdKO axons are characterized by enhanced localization of glycolytic enzymes. (A) Schematic representation of the glycolytic pathway. Glucose is first processed consuming ATP in the preparatory phase, of which PFKP is rate-limiting. During the pay-off phase, per molecule of glucose, two pyruvate molecules are generated, yielding two ATP per each consumed. The rate of this second half of the pathway is controlled by PKM. Finally, when glycolysis is uncoupled from mitochondrial oxidative phosphorylation, pyruvate is reduced by LDHA to lactate, in what is known as the Warburg effect. (B) Representative images of PFKB immunolabeling at 1 dpi in regenerating axons of both the WT and cdKO genotype. As compared with WT axons, cdKO ones show increased labeling, characterized by bright foci throughout the distal axonal shaft. Scale bar 10 µm. (C) Representative images of PKM immunolabeling (isoform 1 and 2) at 1 dpi in regenerating axons of both the WT and cdKO genotype. Again, cdKO axons reveal a higher degree of labeling as compared with WT ones, with bright foci throughout the distal shaft and at the growth cone. Scale bar 10 µm. (D) Representative images of LDHA immunolabeling at 1 dpi in regenerating axons of both the WT and cdKO genotype. cdKO axons display brighter labeling as compared with WT ones, diffused through the growth cone and the distal shaft of the axon. Scale bar 10 µm. The reporter hSyn-YFP was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp; see Materials and methods. Steps of the pathway that were omitted are represented by a dotted line. F6P (fructose-6-phosphate), F1,6P (fructose-1,6-bisphosphate), and PEP (phosphoenolpyruvate).

Article Snippet: For mitochondrial imaging, cells were transduced with a Cre-dependent AAV2/2-CAG-FLeX-mitoGFP-t2A-mCherry-WPRE vector (Charles River, 2.7 × 10 10 GC/ml final titer in medium).

Techniques: Generated, Immunolabeling, Labeling

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Deletion of Pten and Socs3 increases axotomy-induced axonal regeneration in primary RGCs. (A) Primary retinal cultures are obtained from retinae of Pten fl/fl ;Socs3 fl/fl mice at P2–3 and seeded in microfluidic devices. The RGCs are labeled and conditionally deleted for Pten and Socs3 via AAV2/2-mediated transduction at 1DIV. Axonal injury is performed between 12DIV and 14DIV and the outcome of regeneration is assessed 1 day later (1 dpi, DIV12–14+1). (B) Representative images of uninjured axons (naive), injured axons immediately after axotomy (0 dpi), and regrowing axons (1 dpi) of WT and cdKO RGCs. hSyn-YFP is encoded by an AAV2/2-hSyn-ATeam YEMK -WPRE-hGHp vector (see Materials and methods). Scale bar 100 µm. (C) Representative images of degenerating axons (red), regenerating axons (green), and static axons (orange). The latter survive injury but do not grow past the cut site at 1 dpi and until 3 dpi. Scale bar 50 µm. (D) Quantification of the axons regrowing in the axonal compartment shows that deletion of Pten and Socs3 induces an increase in the percentage of regenerating axons at 1 dpi. (E) Quantification of the axonal length shows that codeletion of Pten and Socs3 induces a significant increase in the average length of regrowing axons at 1 dpi. The median length is reported above both groups. (F) Cumulative distribution of axonal lengths shows that Pten and Socs3 co-deletion increases the length of regrowth at 1 dpi. Data from six independent experiments, presented as mean ± SD (D) or median ± 25–75th confidence interval (E) and bootstrap 95% confidence interval versus WT. Student’s t test (D), Kolmogorov–Smirnov test (E), and Mann–Whitney U test (F). P values are reported within the graphs.

Article Snippet: Only for the cdKO condition, an AAV2/2-hSyn1-Cre-t2A-mKate2 vector (Charles River, 1.8 × 10 9 GC/ml final titer in medium) vector was used in combination with the reporters to induce gene recombination and deletion in Pten fl/fl ; Socs3 fl/fl cells.

Techniques: Labeling, Transduction, Plasmid Preparation, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: AAV-mediated conditional knockout of Pten and Socs3 leads to specific deletion and upregulation of regeneration-associated pathways at 14DIV. (A) Immunolabelling for DAPI, PTEN, and Tuj1 shows specific loss of PTEN within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector (cdKO) as compared to WT cells, indicating specific recombination and gene knockout. Scale bar 50 µm. (B) Quantification of PTEN signal in the somata of hSyn-YFP + RGCs shows that on average 98.7% of the RGCs are positive for PTEN at 14DIV in the WT condition as compared to only 2% in the cdKO culture, indicating that an efficient knockout is achieved. (C) Immunolabeling for DAPI, pS6, and Tuj1 reveals increased phosphorylation of S6 within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector as compared with WT cells, indicating mTOR activation. Scale bar 50 µm. (D) Quantification of pS6 signal in the somata of hSyn-YFP + RGCs at 14DIV shows a ∼30% increase in the number of pS6 + RGCs upon deletion of Pten (P) and Socs3 (S). (E) Immunolabeling for DAPI, pSTAT3, and Tuj1 reveals increased phosphorylation of STAT3 within Tuj1 + /hSyn-YFP + RGCs upon transduction with an AAV2/2-Syn-Cre viral vector as compared to WT cells, indicating JAK/STAT activation. Scale bar 50 µm. (F) Quantification of pSTAT3 signal in the somata of hSyn-YFP + RGCs at 14DIV shows a ∼90% increase in the number of pSTAT3 + RGCs upon deletion of Pten and Socs3 . In all experiments hSyn-YFP was encoded by AAV2/2-hSyn1-ATeam YEMK -WPRE-hGHp ; see Materials and methods. Data from four (A and B) or two (C–F) independent experiments, presented as mean ± SD (B, D, and F) and bootstrap 95% confidence interval versus WT (B, D, and F). Student’s t test (B, D, and F). P values are reported within the graphs.

Article Snippet: Only for the cdKO condition, an AAV2/2-hSyn1-Cre-t2A-mKate2 vector (Charles River, 1.8 × 10 9 GC/ml final titer in medium) vector was used in combination with the reporters to induce gene recombination and deletion in Pten fl/fl ; Socs3 fl/fl cells.

Techniques: Knock-Out, Transduction, Plasmid Preparation, Gene Knockout, Immunolabeling, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Deletion of Pten and Socs3 increases axonal glycolytic energy after injury. (A) Comparison of non-normalized axonal ATP measurements does not identify any significant difference in the intra-axonal concentration of ATP in naive axons between WT and cdKO neurons. (B) Comparison of non-normalized axonal lactate measurements does not identify any significant difference in the intra-axonal concentration of lactate in naive axons between WT and cdKO neurons. (C) Representative images of axons, after PFKP (Phosphofructokinase, Platelet) immunolabeling (magenta), assigned to each different scoring category. Axons with no appreciable labeling are classified as low. Faint labeling is scored as mid, while bright labeling and the presence of foci are scored as high. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp; see Materials and methods. Scale bar 10 µm ( D) Semiquantitative analysis via randomized blind scoring of PFKP immunolabeled axons by two independent evaluators reveals increased labeling in regenerating cdKO axons as compared to WT ones. n > 20 regenerating axons for both genotypes. (E) Representative images for each scoring category of PKM (Pyruvate Kinase M1/2) immunolabeled axons (magenta). Axons with faint signal are classified as low. Labeling with sparse foci is classified as mid, while bright labeling and the presence of abundant foci is scored as high. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp. Scale bar 10 µm. (F) Semiquantitative analysis via randomized blind scoring of PKM immunolabeled axons by two independent evaluators shows increased labeling in regenerating cdKO axons as compared with WT ones. n > 20 regenerating axons for both genotypes. (G) Representative images of axons, after LDHA (Lactate Dehydrogenase A) immunolabeling (magenta), were assigned to each different scoring category. Axons are classified from low to high based on brightness and uniformity of the labeling along the distal end of the axon. Imaging conditions and histogram levels were maintained constant. The reporter hSyn-YFP (green) was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp. Scale bar 10 µm. (H) Semiquantitative analysis via randomized blind scoring by two independent evaluators reveals increased LDHA labeling in regenerating cdKO axons as compared with WT ones. n > 20 regenerating axons for both genotypes. (I) Relative percentage representation of mitochondrial ATP (oligomycin sensitive, green, top) and glycolytic ATP (oligomycin and 2-deoxyglucose sensitive, red, bottom) at 1 dpi. Both static and regenerating cdKO axons show a higher relative percentage of glycolytic ATP as compared to WT axons. The percentage value was obtained by subtracting the baseline measurement in the presence of both 10 µM oligomycin and 50 mM 2-deoxyglucose from the measurements in normal medium and oligomycin-only. Then, the oligomycin-only measurement was expressed as a percentage of the measurement in normal medium. Data from five (A), four (B and I) independent experiments, presented as median ± 25–75th confidence interval (A and B), mean ± SD (D–H), or mean ± SEM (I) and bootstrap 95% confidence interval versus WT (A and B). Mann–Whitney U test (A and B). P values are reported within the graphs.

Article Snippet: Only for the cdKO condition, an AAV2/2-hSyn1-Cre-t2A-mKate2 vector (Charles River, 1.8 × 10 9 GC/ml final titer in medium) vector was used in combination with the reporters to induce gene recombination and deletion in Pten fl/fl ; Socs3 fl/fl cells.

Techniques: Comparison, Concentration Assay, Immunolabeling, Labeling, Imaging, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Local glycolysis supports injury-induced axonal regeneration

doi: 10.1083/jcb.202402133

Figure Lengend Snippet: Regenerating cdKO axons are characterized by enhanced localization of glycolytic enzymes. (A) Schematic representation of the glycolytic pathway. Glucose is first processed consuming ATP in the preparatory phase, of which PFKP is rate-limiting. During the pay-off phase, per molecule of glucose, two pyruvate molecules are generated, yielding two ATP per each consumed. The rate of this second half of the pathway is controlled by PKM. Finally, when glycolysis is uncoupled from mitochondrial oxidative phosphorylation, pyruvate is reduced by LDHA to lactate, in what is known as the Warburg effect. (B) Representative images of PFKB immunolabeling at 1 dpi in regenerating axons of both the WT and cdKO genotype. As compared with WT axons, cdKO ones show increased labeling, characterized by bright foci throughout the distal axonal shaft. Scale bar 10 µm. (C) Representative images of PKM immunolabeling (isoform 1 and 2) at 1 dpi in regenerating axons of both the WT and cdKO genotype. Again, cdKO axons reveal a higher degree of labeling as compared with WT ones, with bright foci throughout the distal shaft and at the growth cone. Scale bar 10 µm. (D) Representative images of LDHA immunolabeling at 1 dpi in regenerating axons of both the WT and cdKO genotype. cdKO axons display brighter labeling as compared with WT ones, diffused through the growth cone and the distal shaft of the axon. Scale bar 10 µm. The reporter hSyn-YFP was encoded by AAV2/2-hSyn1-Laconic-WPRE-hGHp; see Materials and methods. Steps of the pathway that were omitted are represented by a dotted line. F6P (fructose-6-phosphate), F1,6P (fructose-1,6-bisphosphate), and PEP (phosphoenolpyruvate).

Article Snippet: Only for the cdKO condition, an AAV2/2-hSyn1-Cre-t2A-mKate2 vector (Charles River, 1.8 × 10 9 GC/ml final titer in medium) vector was used in combination with the reporters to induce gene recombination and deletion in Pten fl/fl ; Socs3 fl/fl cells.

Techniques: Generated, Immunolabeling, Labeling