Journal: Advanced Science

Article Title: Extracellular Matrix Remodeling Alleviates Memory Deficits in Alzheimer's Disease by Enhancing the Astrocytic Autophagy‐Lysosome Pathway

doi: 10.1002/advs.202400480

Figure Lengend Snippet: ECM remodeling alleviates Aβ pathology and reactivates astrocytes in the mPFC. A) Represent image of 6E10 in mPFC in the denatured ChABC and ChABC groups. The scale bar is 500 µm or 50 µm. B,C) Quantification of the number (B) and diameter (C) of senile plaques in the denatured ChABC and ChABC groups. D) Quantification of the percentage of senile plaques that are greater than or equal to 25 µm, between 10 and 25 µm, and less than or equal to 10 µm in the denatured ChABC and ChABC groups. E) Western blotting analysis of 6E10 in PFC of 5xFAD mice after ChABC or denatured ChABC treatment. F) Immunofluorescent images of GFAP and Iba1 in the mPFC from WT‐denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD‐ChABC mice. The scale bar is 20 µm. G,H) Quantification of the astrocyte cell number (G) and the number of branch (H) in the mPFC from WT‐ denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD‐ChABC mice. I,J) Quantification of the microglial cell number (I) and the number of branch (J) in the mPFC from WT‐ denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD‐ChABC mice. K) Immunofluorescent images of 6E10 (red) and GFAP (green) in mPFC from 5xFAD mice after denatured ChABC or ChABC administration. The scale bar is 20 µm. L) Quantification of the number of astrocytes per plaque in the 5xFAD‐denatured ChABC and 5xFAD‐ChABC mice. M) Quantification of the number of astrocytes from 30 µm, 30 to 60 µm, and 60 to 90 µm around Aβ plaques in the 5xFAD‐denatured ChABC and 5xFAD‐ ChABC mice. N) Colocalization analysis of 6E10 and GFAP from 5xFAD‐denatured ChABC mice (left) and 5xFAD‐ChABC mice (right). Data in (B) – (E), (G) – (J), (L), and (M) are means ± SEM (numbers in bars show cells/ biological replicates). Statistical analyses were performed by unpaired two‐sided Student's t‐test in (B), (C), (E), and (L), Two‐way ANOVA with Bonferroni's multiple comparisons test in (D) and (M), or Brown‐Forsythe and Welch ANOVA with Bonferroni's multiple comparisons test in (G) to (J), * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significant. For additional data, see Figure and .

Article Snippet: A concentration of 1 μg mL −1 FITC‐Aβ 1‐42 and 0.04 U ChABC were added into the cell medium and incubated for 3 h. For lysosome inhibition assay, 100 n m BafA1 (Cat. No. HY‐100558, MCE) was incubated for 12 h before FITC‐Aβ 1‐42 and 0.04 U ChABC administration.

Techniques: Western Blot

Journal: Advanced Science

Article Title: Extracellular Matrix Remodeling Alleviates Memory Deficits in Alzheimer's Disease by Enhancing the Astrocytic Autophagy‐Lysosome Pathway

doi: 10.1002/advs.202400480

Figure Lengend Snippet: ECM remodeling enhances the astrocytic autolysosome pathway and Aβ clearance. A) Western blotting analysis of p62, LC3B I/II, ATG16L1, and pATG16L1 in PFC from WT‐denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD‐ChABC mice. B–D) Quantification of the expression level of p62 (B), LC3BII/LCB3I (C), and pATG16L1/ATG16L1 (D) in the western blotting shown in (A). E) Immunofluorescent images of 6E10 (blue), pATG16L1 (green), and GFAP (red) in mPFC from 5xFAD mice and WT mice after denatured ChABC or ChABC administration. The scale bar is 20 µm. F) Quantification of the fluorescence intensity of pATG16L1 in the WT‐denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD ChABC mice. G) Quantification of the fold of pATG16L1 per astrocytes to total pATG16L1 in the WT‐denatured ChABC, 5xFAD‐denatured ChABC, and 5xFAD‐ ChABC mice. H) Immunofluorescent images of GFAP (green), 6E10 (cyan), EEA1 (red), Rab7 (red), and Lamp1 (red) in mPFC from 5xFAD mice after denatured ChABC or ChABC administration. The scale bar is 20 µm. I–L) Quantification of the colocalization of 6E10 and GFAP (I), 6E10, GFAP and EEA1 (J), 6E10, GFAP, and Rab7(K), and 6E10, GFAP and Lamp1(L) from the 5xFAD mice after ECM remodeling. M) Western blotting analysis of EEA1, Rab7, and Lamp1 from the 5xFAD mice after ECM remodeling. Data in (B) – (D), (F) – (G), and (I) – (M) are means ± SEM (numbers in bars show biological replicates). Statistical analyses were performed by One‐way ANOVA with Bonferroni's multiple comparisons test in (B) to (D) and (F) to (G), unpaired two‐sided Student's t‐test in (I) to (L), or Two‐way ANOVA with Bonferroni's multiple comparisons test in (M), * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significant. For additional data, see Figure and (Supporting Information).

Article Snippet: A concentration of 1 μg mL −1 FITC‐Aβ 1‐42 and 0.04 U ChABC were added into the cell medium and incubated for 3 h. For lysosome inhibition assay, 100 n m BafA1 (Cat. No. HY‐100558, MCE) was incubated for 12 h before FITC‐Aβ 1‐42 and 0.04 U ChABC administration.

Techniques: Western Blot, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Comparing Astrocytic Cell Lines that Are Inhibitory or Permissive for Axon Growth: the Major Axon-Inhibitory Proteoglycan Is NG2

doi: 10.1523/JNEUROSCI.19-20-08778.1999

Figure Lengend Snippet: a, Production of NG2 by A7 and Neu7 cells. In A7 cells, NG2 is not detectable in the supernatant or membrane fraction. It is only detectable in small amounts in the highly concentrated 1 m fraction from ion exchange fractionation. Neu7 produces large amounts of NG2 in cell surface and released forms, seen in the detergent lysate and culture supernatant samples. These samples were digested with chondroitinase ABC. B, NG2 in fractions from anion exchange. The fractions from both A7 and Neu7 coming off the column at 0.5 m NaCl contain NG2, which forms a discrete band in the absence of chondroitinase ABC digestion, and therefore has little or no GAG chain attached. There is a large amount of NG2 in the 1 m fraction from Neu7, but this cannot be seen as a discrete band unless it is digested with chondroitinase ABC, indicating that all the NG2 in this fraction carries GAG chains.

Article Snippet: GAG lyase digestion Samples of 0.05 ml were digested with 0.02 U of protease-free chondroitinase ABC (ChABC) (Boehringer Mannheim) for 2.5 hr at 37°C.

Techniques: Fractionation

Journal: The Journal of Neuroscience

Article Title: Comparing Astrocytic Cell Lines that Are Inhibitory or Permissive for Axon Growth: the Major Axon-Inhibitory Proteoglycan Is NG2

doi: 10.1523/JNEUROSCI.19-20-08778.1999

Figure Lengend Snippet: NG2 Western blot of Neu7 detergent lysate after digestion of samples with various enzymes. No NG2 band at ∼300 kDa is seen in the absence of digestion with chondroitinase ABC or AC, but after digestion with these enzymes, a sharp band is seen. Chondroitinase ABC and AC produce the same effect, and there is no additive effect. In Neu7 detergent lysate, a larger unidentified band at higher molecular weight is also seen. On digestion with keratanase, the 300 kDa band disappears, and a new band is seen at 100 kDa. When chondroitinase ABC is added to the keratanase, a 200 kDa band is seen in addition to the 100 kDa band. Digestion overnight with keratanase and chondroitinase (right lane) leads to almost complete disappearance of the 200 kDa band.

Article Snippet: GAG lyase digestion Samples of 0.05 ml were digested with 0.02 U of protease-free chondroitinase ABC (ChABC) (Boehringer Mannheim) for 2.5 hr at 37°C.

Techniques: Western Blot, Molecular Weight