Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A – D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with 100 μM NMDA and its co-agonist 10 μM glycine (hereafter referred to as ‘NMDA’). Immunofluorescence ( A ) and immunoblotting ( B ) used a C-terminal (C-ter) isoform-specific antibody (TrkB-FL Ct) recognizing both the full-length protein (FL) and the intracellular fragment (f32). A Shows TrkB-FL (green) and nuclei (blue, DAPI stain). Arrowheads indicate varicosities in neuronal projections. Scale bar: 20 μm. Insets show cell body details for untreated cells and cells treated with NMDA for 120 min. B Compares the decrease in TrkB-FL and formation of f32 with PSD-95 downregulation, detected using a C-terminal antibody (PSD-95 Ct). Calpain activation was confirmed by the accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) served as a loading control for protein normalization. C , D Quantification of normalized TrkB-FL and PSD-95 levels, shown relative to levels in the absence of NMDA (control). Data are represented as means ± SD. Statistical analysis: one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (** P < 0.01, *** P < 0.001, **** P < 0.0001; 0-90 min, n = 5; 120 min, n = 3). E , F Effect of dynasore preincubation (80 μM, 30 min) on TrkB-FL levels after 2 h NMDA treatment. Data are means ± SD ( n = 4); statistical analysis as above (* P < 0.05). G Sequences of the cell-penetrating neuroprotective peptide (MTFL 457 ) and control peptide (MTMyc). Both contain Tat amino acids (aa) 47–57 (italic), followed by rat TrkB-FL aa 457-471 (light blue) or c-Myc aa 408-421 (dark blue), respectively. H , I Effect of peptide preincubation (25 μM, 30 min) on TrkB-FL shedding and processing during NMDA treatment. Culture media were analyzed using an antibody against the TrkB-FL extracellular domain (panTrkB), which recognizes the ectodomains (ECDs) of all isoforms (TrkB-ECD), to assess receptor shedding by metalloproteinase (MP) activation. Total lysates were analyzed with the TrkB-FL Ct antibody to evaluate TrkB-FL calpain processing via f32 production. Relative TrkB-ECD levels are shown as means ± SD (0-4 h, n = 7; 6 h, n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test (** P < 0.01, **** P < 0.0001, comparing each peptide + NMDA vs. peptide alone; # P < 0.05, #### P < 0.0001, comparing MTMyc vs. MTFL 457 at each time point). J , K Effect of NMDA on total and cell-surface pY816-TrkB-FL levels. Cultures were preincubated with peptides as above, then treated briefly with NMDA (1 h) to minimize receptor degradation. Cell-surface proteins were biotin-labeled and precipitated, then compared to corresponding total lysates. Data are represented as means ± SD ( n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test ( n.s . = n ot significant; ** P < 0.01, comparing NMDA vs. no NMDA; ## P < 0.01, comparing MTMyc + NMDA vs. MTFL 457 + NMDA).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Western Blot, Staining, Activation Assay, Control, Labeling

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A Comparison of MTFL 457 and MTFL 457 AAA sequences. The latter contains Tat aa 47–57 (italic) followed by the indicated rat TrkB-FL sequence but substituting the highly conserved KFG domain with AAA. B Effect of MTFL 457 AAA on neuronal viability in cultures preincubated with MTMyc, MTFL 457 or MTFL 457 AAA as before, and treated with NMDA (2 h). For each peptide, results obtained in excitotoxic conditions were expressed relative to those obtained in the absence of NMDA. Five completely independent experiments were carried out, each including sample triplicates for every condition tested. Means ± SD are represented, and statistical analysis was performed by two-way ANOVA followed by a Bonferroni test (*** P < 0.001 for NMDA effect on MTMyc-treated cells; # P < 0.05 for MTFL 457 vs. MTMyc effect on NMDA-treated cells). C Timeline to analyze in vivo effects of MTMyc, MTFL 457 or MTFL 457 AAA in the model of ischemia. Microvascular photothrombotic permanent damage was initiated in mice by cold-light irradiation (10 min) of a stereotaxically selected brain area after i.v . injection of photosensitive dye Rose Bengal as detailed in Materials and methods. CPPs (10 nmol/g) were retro-orbitally administered 10 min after damage initiation, and animals were sacrificed 24 h or 72 h later. D Representative 1 mm brain coronal slices stained with TTC corresponding to animals injected with MTMyc, MTFL 457 or MTFL 457 AAA and sacrificed at 24 h. E and F Infarct volume of CPP-injected animals expressed as a percentage of the contralateral hemisphere volume. Means ± SD are given for animals sacrificed 24 h ( E ; n = 9-12) or 72 h ( F ; n = 7-10) after damage initiation. Infarct volumes for MTFL 457 and MTFL 457 AAA experimental groups are also expressed as a percentage of values obtained in animals injected with MTMyc. G , H Evaluation of balance and motor coordination. The number of contralateral hind paw slips was measured in animals 24 h ( G , n = 9-17) or 72 h ( H , n = 7-10) after damage induction. E – H Differences with control animals (MTMyc group) were analyzed by Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Comparison, Sequencing, In Vivo, Irradiation, Injection, Staining, Control

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A – C Analysis by immunofluorescence of TrkB-FL/Hrs colocalization. A Cortical neurons were treated with NMDA for the indicated times and analyzed with antibodies specific for Hrs (green) and TrkB-FL Ct (red); nuclear staining was performed with DAPI (blue). Representative images obtained by confocal microscopy correspond to single sections and show the fused channels. Scale bar: 20 μm. B Mean values ± SD of Pearson correlation coefficient (PCC; n = 3). For each independent experiment, a minimum of 80 different neurons were analyzed. Statistical analysis was performed using a generalized linear model followed by a post-hoc Fisher’s LSD test (* P < 0.05, compared to untreated cultures). C Percentage of cells showing at different times of NMDA treatment a PCC ≥ 0.50, generally considered as a threshold for protein colocalization. Statistical analysis was performed as before ( * P < 0.05, compared to untreated cultures; n = 3). D , E Analysis by immunoprecipitation of TrkB-FL/Hrs interaction. Neuronal cultures were treated with NMDA (100 μM) or BDNF (100 ng/ml) for 30 min and compared to untreated cultures. Immunoprecipitation was performed with the Hrs antibody, and the immunoprecipitated proteins (IP) were analyzed by immunoblot using the same antibody ( D ) or TrkB-FL Ct ( E ). Total protein lysates were analyzed in parallel with the immunoprecipitated proteins. Mean values ± SD ( n = 5, except for BDNF-treated cells where n = 4) of Hrs and TrkB-FL levels in NMDA- or BDNF-treated cultures relative to untreated cells are represented for both total lysates and Hrs-immunoprecipitated proteins. Statistical analysis was performed as above ( *P < 0.05, compared to the respective untreated cultures; # P < 0.05 and ## P < 0.01, as indicated).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A Cortical neurons were preincubated with MTMyc and MTFL 457 (25 μM, 30 min) and treated with NMDA for the indicated times. Cells were analyzed by immunofluorescence with antibodies for Hrs (green) and TrkB-FL Ct (red), together with DAPI staining (blue). Representative confocal microscopy images correspond to single sections and show the fused channels. Scale bar: 10 μm. B Mean values ± SD of Pearson correlation coefficient (PCC; n = 3). For each independent experiment, a minimum of 80 different neurons were analyzed. Statistical analysis was performed using a generalized linear model followed by a post-hoc Fisher’s LSD test ( P = 0.06, 0 vs. 60 min of NMDA treatment in MTMyc-cultures). C , D Analysis by immunoprecipitation of TrkB-FL/Hrs interaction. Cultures preincubated with cell-penetrating peptides (CPPs) as above were treated with NMDA for 30 min and compared to untreated cultures. Immunoprecipitation (IP) was performed with the Hrs antibody, and the immunoprecipitated proteins were analyzed by immunoblot using the same antibody ( C ) or TrkB-FL Ct ( D ). Total protein lysates and immunoprecipitated proteins were analyzed in parallel. Mean values ± SD ( n = 4) of Hrs and TrkB-FL levels relative to those found in cells preincubated with MTMyc and without NMDA are represented. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni test (* P < 0.05, ** P < 0.01).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: Cortical neurons were treated with NMDA for the indicated times and analyzed by immunofluorescence with antibodies specific for the Golgi matrix protein GM130 (green) and TrkB-FL Ct (red); nuclear staining was performed with DAPI (blue). Excitotoxicity was induced in the absence of peptides ( A – C ) or after preincubation with MTMyc and MTFL 457 (25 μM, 30 min) ( D – F ). A , D Representative images obtained by confocal microscopy corresponding to single sections, showing channels fused. Scale bar: 20 μm. B , E Mean PCC values ± SD (0-30 min, n = 4; 60 min, n = 3) for TrkB-FL and GM130 colocalization. Statistical analysis was performed using a generalized linear model followed by a post-hoc Fisher’s LSD test (* P < 0.05, ** P < 0.01, compared to basal conditions without peptide or with MTMyc, respectively). C , F Percentage of cells showing, at different treatment times, a PCC ≥ 0.52 ( C ), the value obtained for TrkB-FL/GM130 colocalization in basal conditions in the absence of peptide, or ≥ 0.50 ( F ). Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test (* P < 0.05, compared to basal conditions without peptide or with MTMyc, respectively; 0–30 min, n = 4; 60 min, n = 3). For each independent experiment, a minimum of 80 different neurons were analyzed.

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A Strong association between neuronal degeneration and serum protein leakage after ischemic damage. Immunohistochemistry of brain coronal sections from animals sacrificed 5 h after insult was performed with an antibody recognizing a calpain-generated neoepitope in spectrin N-terminal fragment (SNTF, magenta), labeling cells where this protease is overactive, and a mouse antibody recognizing GM130 (red). Neurodegeneration was also detected by Fluoro-Jade C (FJC) staining (green). Three different tissue areas were compared: the ischemic core, an area peripheral to the infarct core, and the equivalent area of the contralateral hemisphere. Leakage of mouse immunoglobulins due to early blood-brain barrier (BBB) breakage after the ischemic insult, detected by the secondary anti-mouse antibody (see Fig. S ), was observed in the neurodegenerating tissue and strongly interfered with GM130 detection. B , C GM130 staining after preincubation of coronal sections with an anti-mouse IgG (Fab specific) antibody to improve detection. Animals were retro-orbitally injected with peptides MTMyc or MTFL 457 (10 nmol/g) 10 min after damage initiation and sacrificed 5 h later. Comparison of the contralateral ( B ) and the ischemic peripheral areas ( C ). Representative images correspond to single sections. Scale bar: 10 µm. D Model of TrkB-FL regulation in excitotoxicity and MTFL 457 action. Endocytosis of neurotrophin receptor TrkB-FL is promoted by excitotoxicity in neurons treated with control peptide MTMyc or without treatment (left panel). In endosomes, TrkB-FL interacts with the protein Hrs and is retrogradely transported to the Golgi apparatus (GA), where activation of organelle-associated proteinases would be responsible for receptor processing by calpain and regulated intramembrane proteolysis (RIP). Although partial recycling back to the membrane might occur via mechanisms similar to those found after BDNF activation, there is a strong decrease in BDNF/TrkB-FL signaling and CREB/MEF2 promoter activities, causing transcriptional changes that favor neuronal death. In parallel, the GA is disrupted, a hallmark common to many neurodegenerative diseases (NDDs). The neuroprotective peptide MTFL 457 interferes with the TrkB-FL/Hrs interaction induced by excitotoxicity, receptor retrograde transport and processing, as well as GA fragmentation (right panel). We propose that interference by MTFL 457 with the TrkB-FL/Hrs interaction might favor rapid recycling back to the membrane, similar to that of isoform TrkB-T1, sustained BDNF/TrkB-FL/PLCγ signaling, and promotion of neuronal survival.

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunohistochemistry, Generated, Labeling, Staining, Injection, Comparison, Control, Activation Assay, Membrane