Fig 2: Drp1 hyperactivation in mature OLs of AD models.(A) Total protein lysates were harvested from the frozen postmortem cortex of AD patients and control subjects (Table 2). Western blot analysis was performed in the absence of 2-mercaptoethanol (ß-ME) to examine Drp1 oligomers. Histogram: the relative density of Drp1 tetramer versus Drp1 monomer is shown. Student’s t test. (B) Postmortem cortex sections from normal subjects and AD patients (Table 1) were stained with anti–p-Drp1 Ser616 and anti-CC1 antibodies. Scale bar, 50 µm. The number of p-Drp1 Ser616+ CC1+ cells per square millimeter was quantified and is presented in the histogram. Student’s t test. (C) Total protein lysates from the corpus callosum of WT and 5XFAD mouse brains were harvested at the ages of 3 and 6 months. Drp1 tetramer was analyzed by Western blot analysis in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. n = 3 mice per group. Student’s t test. (D) Mature OLs were differentiated from OPCs and exposed to oligomeric Aß1-42 for the indicated periods. Drp1 tetramer was analyzed by Western blot analysis of total protein lysates in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. The data from five independent experiments were analyzed using one-way ANOVA with Tukey’s post hoc test. (E) Mature OLs were treated with oligomeric Aß1-42 for 24 hours and stained with anti–p-Drp1 Ser616 (green) and anti-MBP (red) antibodies. The intensity of p-Drp1Ser616 in the MBP+OLs was quantitated and is presented in the histogram. The data from three independent experiments were compared using the Student’s t test. Scale bar, 20 µm. (F) A linear correlation between the relative Drp1 tetramer density and the cleavage of GSDMD is shown. All data are expressed as the mean ± SEM.

Fig 3: Microglial process convergence onto injured axonal swellings occurs 1 day following cFPI in the micro pig. Representative reconstructions of (A,B) MBP+ intact myelinated axons (red) or (C,D) APP+ injured axonal swellings (green) and Iba-1+ microglia (white) in the thalamus of injured micro pigs 1 day post-cFPI. DAPI-labeled nuclei are in blue. Bar graphs depicting the average number of (E) total microglial process contacts, (F) terminal processes, (G) process cups and (H) process passes of Iba-1+ microglial that contact either MBP+ intact fibers or APP+ injured axonal swellings 1 day following TBI. As previously seen, total contacts between microglial processes and injured axonal swellings was increased in the micro pig thalamus 1 day following cFPI. While there were not differences between intact and injured axonal segments in regards to microglial process passes or process cups, the increase in total process contacts was mirrored in the number of terminal processes contacting injured axonal swellings. Graphs depicts the mean ± standard error of the mean. *p < 0.05. Scale bar: 5 µm.

Fig 4: Mature OL-specific Drp1 heterozygous knockout is protective in 5XFAD mice.(A) Brain sections containing the corpus callosum from 6-month-old 5XFADhet;Drp1+/+ and 5XFADhet;CNP;Drp1fl/+ mice were stained with anti-GSDMD, anti-MBP, and anti-6E10 antibodies. (B) The colocalization of GSDMD and Aß plaques (top) and the localization of GSDMD in MBP+OLs (middle) were quantified. The intensity of GSDMD in the Aß plaque area was analyzed (lower). n = 3 to 4 mice per group. (C) Brain sections from 6-month-old WT, CNP;Drp1fl/+, 5XFADhet;Drp1+/+, and 5XFADhet;CNP;Drp1fl/+ mice were stained with anti–IL-1ß (green) and anti-CC1 (red) antibody for immunofluorescence analysis. n = 4 mice per group. Scale bar, 20 µm. (D) The intensity of IL-1ß in CC1+OLs was quantified. (E) Mouse brain sections were stained with the Black Gold II myelin staining kit (top) and anti-MBP for immunohistochemistry analysis (bottom). Scale bar, 200 µm. (F) The area of positive anti-MBP staining was quantified and is presented in the histogram. (G) The corpus callosum of each mouse of the indicated genotypes was harvested for TEM analysis. (H) The g-ratio of the myelin of the mice was measured. (I) The percentage of myelinated axons was quantified. (J) Cognitive activity was assessed in 6-month-old mice using the Y-maze. Nine female and 10 male WT, 8 female and 8 male CNP;Drp1fl/+, 9 female and 10 male 5XFADhet;Drp1+/+, and 7 female and 6 male 5XFADhet;CNP;Drp1fl/+ mice were used. (K) Movement activity was assessed in 6-month-old mice using the open-field chamber. The total travel distance of mice is shown. Six female and 6 male WT, 8 female and 7 male CNP;Drp1fl/+, 8 female and 8 male 5XFADhet;Drp1+/+, and 7 female and 7 male 5XFADhet;CNP;Drp1fl/+ mice were used. All the data are expressed as the mean ± SEM and compared by one-way ANOVA with Tukey’s post hoc test.

Fig 5: Microglial processes are less engaged with axons following cFPI in the rat. Representative 3D reconstructions of Iba-1+ microglial (white) processes with (A) MBP+ intact myelinated axons (red) in sham-injured rats or APP+ injured axonal swellings (green) in cFPI rats either (B) 6 h or (C) 1 day post-injury. DAPI-labeled nuclei are in blue. Bar graphs depicting the average number of (D) total microglial process contacts, (E) terminal processes, (F) process cups, and (G) process passes of Iba-1+ microglial that contact either MBP+ intact fibers or APP+ injured axonal swellings following sham or TBI in the rat. Note that overall, total microglial process contacts are decreased in intact axonal segments at both 6 h and 1 day post-cFPI and in injured axonal swellings at 6 h post-injury in rats. Graphs depict the mean ± standard error of the mean. *p < 0.05 compared to sham; #p < 0.05 compared to injured axonal swellings 1 day post-cFPI; &p < 0.05 compared to intact axonal segments 6 h post-cFPI. Scale bar: 5 µm.