Fig 2: Specificity of Cx3cr1-Cre lines following SE. A, B, Full montage of confocal images showing GFP reporter expression in the CTX and hippocampus of Cx3cr1-Cre±;Rosa-Greenf/- mice 3 d post-SE induced by pilocarpine. Mouse brain sections were stained with goat anti-Iba1 (pink), rabbit anti-NeuN (red) or chicken anti-GFAP (red), and DAPI (blue). Scale bar, 200 μm. C, D, High-magnification images from CTX, CA1, CA3, and DG. Scale bar, 20 μm. E–G, Full montage of confocal images showing GFP reporter expression in the CTX and hippocampus of Cx3cr1-Cre±;mT/mGf/- mice 3 d post-SE induced by pilocarpine. Mouse brain sections were stained with rabbit anti-GFP, goat anti-Iba1 (pink) or mouse anti-NeuN or chicken anti-GFAP, and DAPI (blue). Scale bar, 200 μm. H–J, High-magnification images from CTX, CA1, CA3, and DG. Scale bar, 20 μm. Also see Extended Data Fig. 1-3.

Fig 3: Fucoxanthin activated autophagy after TBI.(A) Representative images of immunofluorescence for LC3 surrounding the injured cortex. LC3 punctate dots were observed in the cytoplasm by immunofluorescent staining of LC3 (red). Neuron cells and nuclei are labeled with NeuN (green) and DAPI (blue), respectively. Magnification: 40 x. Scale bar: 50 mm. (B) Mice brain tissues were collected 1 day after TBI in different groups, and the expression of LC3, Beclin-1 and p62 was measured by western blot. Fucoxanthin treatment significantly increased the level of LC3-II and Beclin-1 while decreasing the level of p62 after TBI. (C) 3-MA (400 nM) was injected i.c.v. 30 min before TBI. Mice were then subjected to TBI and treatment of fucoxanthin 30 min after TBI. Pretreatment with 3-MA significantly attenuated fucoxanthin-induced activation of autophagy and suppression of apoptosis and oxidative stress in the ipsilateral cortex. Data are presented as mean ± SEM, n = 6 per group; **p < 0.01, ***p < 0.001 versus sham group; #p < 0.05, ##p < 0.01 versus TBI + vehicle group; &&p < 0.01, &&&p < 0.001 versus TBI + fucoxanthin group. β-actin was used as a loading control.

Fig 4: In vivo d(X)TAT protein delivery targets both neurons and astrocytes.(A) Analysis of cell density in the area of highest tdTom fluorescence intensity, defined as region 1 in Fig. 3B. DAPI is used to stain all cells, while GFAP and Iba1 are used as markers of astrocytes and microglia cells, respectively. Brain tissue injected with d(WW)TAT/TAT-Cre is compared to AAV2-Cre injections and noninjected tissue. The data presented are the average cell densities in 200 μm–by–200 μm areas from injection slices obtained from two different animals. No statistical significance was seen between regions for each type of marker. (B) Identification and quantification of the cells edited by Cre in region 2 (as defined in Fig. 3B). Brain slices immunostained for markers of neurons (NeuN), astrocytes (GFAP), and microglia (Iba1) were imaged for colocalization with tdTom by fluorescence microscopy. Representative images are provided for TAT-Cre/d(WW)TAT injections, with tdTom pseudocolored red and NeuN and GFAP stains pseudocolored green. Colocalization of cells positive for tdTom fluorescence and cell-specific markers was used to determine the percentage of neurons and astrocytes successfully gene edited within region 2. Conversely, the transduction efficiency, using tdTom expression as a proxy for Cre delivery, was assessed by measuring the percentage of tdTom cells among all cells stained for cell-specific markers. The data represent the averages obtained from N = 3 animals per group, with each injection being imaged at a minimum of five distinct locations showing tdTom fluorescence. Coordinates are overlaid in each panel to indicate the direction of the images in relation to the center of intensities.

Fig 5: Treatment with bpV(pic) spares MNs in both in vivo and in vitro models of ALS. A The treatment schedule and experimental design for in vivo therapy of mSOD1G93A ALS mice. B and D Ventral horn motor neurons were significantly less than wild-type (WT) mice at 90 days of age (p < 0.001). A late pre-symptomatic daily treatment schedule between days 70 and 90 of age enhanced the number of NeuN-positive motor neurons (p = 0.003) but did not significantly alter Iba-1-positive microglial reactivity (p = 0.701) in the ventral horn of the lumbar spinal cord in mSOD1G93A mice compared to vehicle-treated mice (C and D). E bpV(pic) improved morphology and significantly enhanced viability (p = 0.018) and reduced LDH release (p = 0.025) in 24-h serum-starved mSOD1G93A-expressing motor neuron cells. F) In addition, bpV(pic) significantly increased p-AktS473/Akt ratio following treatment (p < 0.05) while co-culture with LY294002 under starvation conditions blocked bpV(pic)’s stimulating effects (p < 0.01). G & H) Gastrocnemius muscle assessment showed NMJs were innervated significantly more in bpV(pic)-treated mice than vehicle control mice (p = 0.018), but the treatment did not significantly affect muscle size and weight. ALS amyotrophic lateral sclerosis (ALS); NeuN neuronal nuclear antigen; mSOD1G93A Mutant superoxide dismutase 1 G93A; LDH lactate dehydrogenase; NMJ neuromuscular junction. WT group, n = 6; vehicle and bpV(pic) groups, n = 7 each). Scale bars = 250 μM (B and C) and 50 μM (H). All cell culture experiment data are from experiments performed in triplicate