Journal: iScience
Article Title: Suppressing phagocyte activation by overexpressing the phosphatidylserine lipase ABHD12 preserves sarmopathic nerves
doi: 10.1016/j.isci.2025.112626
Figure Lengend Snippet: In vivo neuronal ABHD12 overexpression in sarmopathy mouse model reduces macrophage activation and rescues motor axon loss and neuropathy (A-A′) Representative images of AAV-mediated, neuron-specific expression of EGFP in the distal femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice. EGFP expression reveals a population of blebbing axons. (B) Representative images of CD68 immunostaining and EGFP-labeled axons in femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice. (C) Schematic for macrophage-axon association quantification. (D) Macrophage-axon distance quantification in femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice ( n = 3 animals, each point replicates a counted axon). (E) Schematic of AAV-mediated, neuron-specific expression of EGFP or ABHD12-EGFP in 1-month-old Nmnat2 V98M/R232Q sarmopathy mice. (F and G) Representative images of CD68 immunostaining in femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice after two months of control (EGFP) or Abhd12 gene therapy. (H) Representative images of Iba1 and CD68 immunostaining in femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice after two months of Abhd12 gene therapy. (I) Quantification of activated CD68 + and total Iba1+ macrophages in femoral nerve of 3-month-old Nmnat2 V98M/R232Q sarmopathy mice after two months of control (EGFP) or Abhd12 gene therapy. (J) Percent axonal area/total nerve area for femoral nerve calculated at 3months (2months of AAV-mediated gene therapy with control ( n = 4) or Abhd12 IgG ( n = 5). (K) Average time suspended from an inverted screen (max. 120 s) for EGFP control ( n = 10) and ABHD12 gene therapy Nmnat2 V98M/R232Q ( n = 14) mice at 2 and 3 months of age. All data are presented as mean ± SEM. Statistical significance determined by Student’s unpaired t test or one-way ANOVA with multiple comparisons. ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Article Snippet: Six μm–thick sections of sciatic nerves were prepared on a cryostat (Leica CM1860), mounted onto slides, and processed as previously described using the following antibodies: (a) for activated macrophages , primary Ab CD68 (1:100, Bio-Rad, MCA1957GA) and secondary Ab anti-rat Cy3 (1:500, Jackson Immunoresearch, 112-165-143); (b) for total macrophages , primary Ab Iba-1 (1:500, Wako Chemicals, 019–19741) and secondary Ab anti-rabbit Alexa Fluor 488 (1:500, Invitrogen, A11034); Sciatic nerves were imaged with the Leica DMI 4000B confocal microscope at ×20 magnification.
Techniques: In Vivo, Over Expression, Activation Assay, Expressing, Immunostaining, Labeling, Control
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