vectabond  (Vector Laboratories)

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Axon amyloidosis and enlargement are important pathological phenotypes in AD. (A) Axon amyloidosis was detected in AD frontal lobe tissues with the Aβ antibody with green fluorescence (Alexa Fluor 488). (A1) Aβ expression was detected in both a senile plaque (indicated by an asterisk) and axons (indicated by arrows). (A2) An axon at the longitudinal orientation showed spheroid formations with Aβ expression (indicated by an arrow). (A3) Two clusters of Aβ-positive axons in the longitudinal direction (indicated by dashed lines) were observed, indicating Aβ staining of axon bundles. An Aβ-containing neural cell is indicated by a red arrow. (A4) A cluster of Aβ-positive axons in the longitudinal direction (indicated by dashed lines) was detected. Blood vessel lumen Aβ expression is indicated by a red arrow. An axon with clear Aβ-staining is indicated by a yellow arrow. (A5) Two clusters of Aβ-positive axons in the transverse direction (indicated by dashed lines) were observed. Individual axons are indicated by yellow arrows. (A6) Axons with abundant axonal Aβ staining (mostly in the transverse direction; yellow arrows) were observed in the white matter. One longitudinal axon is indicated by a red arrow. Scale bar: 50 μm. (B) A general enlargement of axons in AD brain tissues was observed when comparing control axons (indicated by white arrows, top panel) marked by MAP2 antibody staining (green, Alexa Fluor 488) and amyloid-loaded axons in AD (indicated by yellow arrows, bottom two panels) marked by Aβ immunostaining (green, Alexa Fluor 488). A senile plaque in the bottom panel is labeled with an asterisk. Scale bar: 50 μm. (C) Imaging measurements showed that Aβ-positive axons ( n = 200) were 1.72 ± 0.52 fold larger than control axons ( n = 34; * P < 0.001, Mann–Whitney U test). AD: Alzheimer’s disease; Aβ: amyloid-β; Map2: microtubule associated protein 2.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Fluorescence, Expressing, Staining, Control, Immunostaining, Labeling, Imaging, MANN-WHITNEY

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Axons in AD frontal brain tissues show a decrease in axonal structural protein MAP2. (A) At longitudinal (top panel) and transverse orientations (bottom panel), significantly fewer MAP2-positive axons were observed in AD brain tissues compared with control brain tissues. The top panel shows that there were 26 long MAP2-positive axon fragments (representative axons are indicated by white arrows) in the control sample and only 12 long MAP2-positive axon fragments (indicated by yellow arrows) in the AD sample, indicating 53.8% fewer MAP2-positive axons in AD patient samples. The bottom panel shows that there were 391 strong MAP2-positive axon dots (those with an axon bundle formation are indicated by white dashed lines) in the control frontal lobe tissue sample and 79 strong MAP2-positive axon dots (representative dots are indicated by yellow arrows) in the AD frontal lobe tissue sample, which indicates 79.8% fewer MAP2-positive axon signals in AD samples. Scale bar: 100 μm. (B) Three representative images show the correlation between the decrease in axonal MAP2 signals and the increase in Aβ immunostaining in axons of AD brain tissues. The top two images show axons in the gray matter with greatly reduced MAP2 staining (Aβ-laden axons are indicated by yellow arrows and the unaffected axons are indicated by white arrows). The yellow dashed lines in the second image surround a dense-core senile plaque with barely detectable MAP2 immunostaining. The bottom image shows numerous Aβ-loaded axons with diminished MAP2 staining in the white matter. Scale bar: 50 μm. (C) Two representative images show greatly reduced axonal MAP2 staining in Aβ-laden axons labeled with amyloid blue autofluorescence in the white matter. The top panel shows axons primarily in the longitudinal direction, and the bottom panel shows axons primarily in the transverse direction. The yellow arrows indicate the Aβ-loaded axons and the white arrows indicate the control axons without blue autofluorescence. Axons with amyloid blue autofluorescence showed much weaker MAP2 intensity (0.36 ± 0.07, n = 100) compared with axons without amyloid blue autofluorescence ( n = 25). Scale bar: 50 μm. AD: Alzheimer’s disease; Aβ: amyloid-β; Map2: microtubule associated protein 2.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Control, Immunostaining, Staining, Labeling

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Lysosomal destabilization is common in AD frontal lobe tissues. (A–C) Three representative plaques associated with lysosome destabilization. The top plaque was a typical dense-core plaque, whereas the bottom two plaques were more diffuse. All three plaques were associated with enlarged and diffuse Cathepsin D staining (red, Alexa Fluor 594) patterns (yellow dashed lines or yellow arrows), which is a characteristic of lysosome destabilization and lysosome permeabilization that is in contrast to the typical, granule-like strong lysosome staining with focal Cathepsin D positivity (indicated by white arrows in the top panel). Quantitation showed that 71.2% ± 9.6% ( n = 10) of Cathepsin D staining in the senile plaque regions located in lysosomal compartments displayed abnormal diffusive patterns. Weak Aβ staining (green, Alexa Fluor 488) was observed in these abnormal lysosomal compartments. (D–F) Three representative images illustrating diffuse Cathepsin D staining (red, Alexa Fluor 594) in the Aβ-positive axons (green, Alexa Fluor 488; indicated by yellow arrows). Simultaneously, colocalization between Aβ and Cathepsin D in lysosomes was abundant (indicated by white arrows). These three images were acquired using longer exposure settings than other images to show the weak axonal Aβ and Cathepsin D staining. (G) Intracellular lysosomes distant from amyloid plaques showed mostly clear, strong, and granule-like lysosomal Cathepsin D staining (red, Alexa Fluor 594, indicated by white arrows). Scale bar: 50 μm. AD: Alzheimer’s disease; Aβ: amyloid-β.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Staining, Quantitation Assay

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Lysosomal destabilization is strongly associated with Aβ in the cell bodies of neural cells in AD frontal lobe tissues. (A) A control neural cell with little Aβ staining (red, Alexa Fluor 594) showed mostly normal lysosome Cathepsin D staining (green, Alexa Fluor 488) patterns (indicated by white arrows). (B, C) Intracellular Aβ (red, Alexa Fluor 594) was associated with lysosome destabilization, which could be identified at the single lysosome level by significant lysosome enlargement and diffuse staining (indicated by yellow arrows), whereas control lysosomes (indicated by white arrows) showed compact and bright Cathepsin D staining (green, Alexa Fluor 488). (D, E) Two representative images indicating that intracellular Aβ expression was associated with lysosome enlargement, clustering, and diffuse staining. The dashed boxes highlight the areas enriched for both Aβ staining and lysosomal destabilization. (F, G) Two representative images of neural cells containing not only lysosomal Aβ (red, Alexa Fluor 594) and Cathepsin D (green, Alexa Fluor 488) but also extra-lysosomal cytoplasmic Aβ, nuclear Aβ, and nuclear Cathepsin D. Vertical arrows indicate cytoplasmic Aβ staining outside of Cathepsin D-stained areas and horizontal arrows indicate nuclear Aβ and Cathepsin D staining. Scale bar: 25 μm. AD: Alzheimer’s disease; Aβ: amyloid-β.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Control, Staining, Expressing

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Blood and vascular proteins co-exist with intracellular Aβ in neural cells in AD frontal lobe tissues and may also be related to lysosome instability. (A) Representative images showing the co-expression of blood-related markers HBA (green, Alexa Fluor 488) and Aβ (red, Alexa Fluor 594). (B) Representative images showing the co-expression of vascular markers ACTA2 (green, Alexa Fluor 488) and Aβ (red, Alexa Fluor 594). (C) Representative images showing the co-expression of vascular markers ColIV (red, Alexa Fluor 594) and Aβ (green, Alexa Fluor 488). The colocalization of markers is indicated by yellow arrows. Two small blood vessels are indicated by asterisks in the ColIV staining panel. (D) Intracellular HBA (green, Alexa Fluor 488) expression domains overlapped with the region of lysosomal destabilization, which is indicated by enlarged and clustered lysosomal compartments and diffuse Cathepsin D staining (red, Alexa Fluor 594, marked with yellow dashed circles). Control HBA-unaffected lysosomes are indicated by white arrows. Scale bars: 25 μm. ACTA2: Smooth muscle actin alpha 2; AD: Alzheimer’s disease; Aβ: amyloid-β; ColIV: collagen type IV; HBA: hemoglobin.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Expressing, Staining, Control

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Destabilized lysosomes are located in AD frontal lobe tissue dystrophic neurites, tangles, axons, and neuropil threads, and there was co-distribution between phos-tau and Aβ in dystrophic neurites, tangles, neuropil threads, and axons. (A) Abnormal lysosome Lamp2 staining (red, Alexa Fluor 594; indicated by yellow arrows) was detected in peri-plaque dystrophic neurites (top two panels), a representative tangle (third panel), neuropil threads (fourth panel), a representative longitudinal axon (fifth panel) and white matter axons (mostly in the transverse direction; bottom panel) marked with phos-tau immunostaining (green, Alexa Fluor 488). We observed long, diffuse, but relatively weak Lamp2 staining along the length of axons and neuropil threads, which highlights the intricacy of this defect. The bottom three images were acquired using longer exposure time settings to show the relatively weak signals in neuropil threads and axons. Control lysosomes not affected by tau phosphorylation are indicated by white arrows. Scale bar: 50 μm. (B) Phos-tau-positive (green, Alexa Fluor 488) dystrophic neurites (yellow arrows, top panel), tangles (yellow arrows), neuropil threads (white arrows; middle panel), and axons (yellow arrows, bottom panel) were also Aβ-positive (red, Alexa Fluor 594). Scale bar: 50 μm. AD: Alzheimer’s disease; Aβ: amyloid-β.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Staining, Immunostaining, Control, Phospho-proteomics

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Axon degeneration in AD bears the markers of hemorrhagic insults in AD frontal lobe tissues. (A–D) Four blood-related markers (ApoE, HBA, HbA1C, and hemin) all showed positivity in enlarged axons labeled with Aβ immunostaining. They were adjacent to senile plaques (top image in each panel) or not associated with senile plaques (bottom image in each panel). ApoE was stained red (Alexa Fluor 594), whereas HBA, HbA1C, and hemin were stained green (Alexa Fluor 488). Aβ was stained green (Alexa Fluor 488) in panel A and red (Alexa Fluor 594) in panels B–D. (E) Degenerating axons were positive for an endosomal/lysosomal marker Sortilin1 (red, Alexa Fluor 594). The affected axons are indicated by arrows and the senile plaques are indicated by asterisks. Scale bars: 50 μm. AD: Alzheimer’s disease; ApoE: apolipoprotein E; Aβ: amyloid-β; HBA: hemoglobin; HbA1C: glycosylated hemoglobin type A1C.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Labeling, Immunostaining, Staining, Marker

Journal: Neural Regeneration Research

Article Title: Pathological axonal enlargement in connection with amyloidosis, lysosome destabilization, and bleeding is a major defect in Alzheimer’s disease

doi: 10.4103/NRR.NRR-D-24-01440

Figure Lengend Snippet: Axonal breakages are occasionally observed in AD frontal lobe tissues. (A) Two broken axons were identified with AGE (green, Alexa Fluor 488) and Aβ (red, Alexa Fluor 594) immunohistochemistry in AD brain tissues. The axonal gap in the top image measured 24.4 μm, whereas in the bottom image, it measured 38.4 μm. The broken regions are marked by dashed lines and the parental axons are indicated by arrows. (B) Two examples of damaged axons associated with large vesicle clusters and spheroid formation enriched for amyloid markers Aβ (red, Alexa Fluor 594) and HbA1C (green, Alexa Fluor 488). The long stretch of clustered vesicles (indicated by dashed lines) in the top panel measured 32.5 μm in length. The maximal width of the two large spheroids (indicated by dashed lines) in the bottom panel reached 6.2 μm (top-right position) and 8.3 μm (bottom-left position), respectively. The affected axons are indicated by arrows. (C) A long axonal region (indicated by dashed lines) with large spheroid formations associated with endosomal/lysosomal domains marked with Sortilin1 immunostaining (red, Alexa Fluor 594). This region measured 26.1 μm in length. The maximal width of the spheroid formation measured 4.1 μm. Scale bars: 50 μm. AD: Alzheimer’s disease; AGE: advanced glycation end products; Aβ: amyloid-β; HbA1C: glycosylated hemoglobin type A1C.

Article Snippet: Frontal lobe paraffin tissue sections of AD patients were purchased from GeneTex (Irvine, CA, USA).

Techniques: Immunohistochemistry, Immunostaining