anti pan ampar  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Ex vivo incubation with EV from monocytes of hyperammonemic rats (HA-M-EV) induces TrkB–NR2B–GluA1–GluA2 pathway activation in hippocampal slices from control rats. Protein content of (A) TrkB (n= 8-18) and membrane expression of (B) TrkB (n= 4-14), (C) NR2B (n= 7-16), (D) NR2A (n= 6-22), (E) GluA2 (n= 5-13), and (F) GluA1 (n= 6-18) in hippocampal slices, assessed by Western blot. For the analysis of membrane expression, the sections were incubated in the presence (+) or absence (−) of the cross-linker BS3. Samples in the absence of BS3 represent the total amount of each protein, whereas samples incubated in the presence of BS3 represent the non-membrane fraction of each protein. Representative images of the blots of each protein are shown. All data are presented as mean ± SEM. Statistical analysis was determined using four separate one-way ANOVAs, each comparing the four baseline groups (C, HA, C-M-EV, and HA-M-EV) with one of the treatment groups (C-M-EV + Forsk, HA-M-EV + PKAi, C-M-EV + TNFα, or HA-M-EV + aTNFα). One-way ANOVA followed by Fisher’s post-hoc tests were performed to compare all groups. Values significantly different from the C group are indicated by asterisk (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), values significantly different from the C-M-EV group are indicated by a (a=p<0.05, aa=p<0.01, aaa=p<0.001, aaaa=p<0.0001), and values significantly different from HA-M-EV group are indicated by b (b=p<0.05, bb=p<0.01, bbb=p<0.001, bbbb=p<0.0001).

Article Snippet: The hippocampal slices were homogenized and analyzed by Western blot as described above, using the following primary antibodies: GluaA1 (Glutamate A1 Subunit of Ampa Receptors, 1:1,000, Millipore, #04-855), GluA2 (Glutamate A2 Subunit of AMPA Receptors, 1:2,000, Proteintech, #11994-1-AP), IL-1R (Interleukin 1-Beta Receptor, 1:500, Abcam, AB106278 ), NR2A (2A subunit of NMDA receptors, 1:1,000, Millipore, #04-901), NR2B (2B subunit of NMDA receptors, 1:1,000, Millipore, #06-600), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (1:500, Abcam, #ab18987), and S1PR2 (1:1,000, Proteintech, #21180-1-AP).

Techniques: Ex Vivo, Incubation, Activation Assay, Control, Membrane, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Schematic representation of the proposed mechanism by which (1) hyperammonemia induces the formation of pathological EV containing TNFR1-TNFα in monocytes, (2) how TNFα and PKA modulate this process, and (3) how these EV induce pathological effects in the hippocampus. Hyperammonemia increases TNF α and activation of its receptor TNFR1 in monocytes, leading to activation of adenylate cyclase and increased cAMP levels and PKA activation. PKA alters both LC3, leading to increased lysosomal pH and impaired expression of lysosomal proteins such as cathepsin-L and LAMP2. This results in lysosomal dysfunction and altered autophagic flux enhances the release from multivesicular bodies (MVBs) of EV containing increased levels of TNFR1 and TNFα. These pathological HA-M-EV enhance activation of the TNFα–TNFR1–S1PR2–IL-1β–BDNF pathway and the TrkB–NR2B–GluA1–GluA2 pathway in hippocampal slices from control rats, inducing neuroinflammation and altered glutamatergic neurotransmission. All these effects are reversed by blocking TNFα with anti-TNFα or inhibiting PKA with H69 in the cultures of monocytes from hyperammonemic rats (green symbols). Activation of TNFR1 with recombinant TNFα (rTNFα) or of adenylate cyclase with forskolin in monocyte cultures from control rats induce essentially the same effects (red arrows). This figure was created using Biorender.com .

Article Snippet: The hippocampal slices were homogenized and analyzed by Western blot as described above, using the following primary antibodies: GluaA1 (Glutamate A1 Subunit of Ampa Receptors, 1:1,000, Millipore, #04-855), GluA2 (Glutamate A2 Subunit of AMPA Receptors, 1:2,000, Proteintech, #11994-1-AP), IL-1R (Interleukin 1-Beta Receptor, 1:500, Abcam, AB106278 ), NR2A (2A subunit of NMDA receptors, 1:1,000, Millipore, #04-901), NR2B (2B subunit of NMDA receptors, 1:1,000, Millipore, #06-600), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (1:500, Abcam, #ab18987), and S1PR2 (1:1,000, Proteintech, #21180-1-AP).

Techniques: Activation Assay, Expressing, Control, Blocking Assay, Recombinant