Fig 2: General description of the approach. (A) Moderate stretch injury upgrades neuronal expression of BACE1 and Aß, downgrades neuronal expression of APP, synaptic abnormalities (e.g., decreased expression of both MAP2 and synaptophysin), and reduced viability. In addition, the injured neurons may secrete Aß debris to injure the healthy neighborhood neurons. The CD206+ microglial cells with normal phagocytic capacity may protect against stretch injury via reducing synaptic abnormalities (I) and phagocytosing the Aß debris (II). The injured microglia (CD16/32+) with reduced phagocytic capacity may injury the healthy neurons via upgrading neuronal expression of BACE1 and Aß, downgrading neuronal expression of APP and downgrading the phagocytic capacity for removal of Aß debris, and synaptic abnormalities (B).

Fig 3: Images of transplanted hiPSC-RGCs in vivo(A and B) Fluorescent images of the flattened whole-mount retina of saline-injected left eye and hiPSC-RGCs injected right eye (B = 4X (scale bar is 750 µm), B’ = 10X (scale bar is 20 µm)).(C–E) Neurite outgrowth of transplanted hiPSC-RGCs taken as two-photon images acquired from live retinal samples. Cryosection images of transplanted hiPSC-RGCs (n = 3) were detected in the GCL and INL layer of the murine retina and co-stained for (F) Brn3, (G) MAP2, and (H) Synapsin I. The scale bar of images is 20 µm.

Fig 4: Dp71 interacts with BRAF35 and colocalizes with BRAF35 and iBRAF during the neuronal differentiation of N1E-115 cells. (A) N1E-115 cell lysates were subject to immunoprecipitation (IP) using anti-BRAF35 antibodies and the immunoprecipitated proteins were analyzed by Western blotting using antibodies against Dp71 and BRAF35. (B) Lysates from N1E-115 cells transiently expressing GFP or GFP-Dp71 were immunoprecipitated using the GFP-Trap system. The captured proteins were subjected to Western blot analysis using antibodies against BRAF35 and GFP. Input corresponds to 5% of protein lysates prior to immunoprecipitation; IgG0, nonspecific antibody; Ub, unbound proteins; B, bound proteins. Undifferentiated (UD) and differentiated (DIF) N1E-115 cells were immunostained with (C) anti-BRAF35 or (D) anti-iBRAF antibodies and anti-Dp71 and were antibody and counterstained with DAPI to visualize nuclei. (E) Undifferentiated and differentiated N1E-115 cells were immunostained for the neuronal differentiation marker MAP2 and counterstained with DAPI to visualize nuclei. Cells were imaged by CLSM, and representative single optical Z-sections are shown (scale bar = 10 µm).

Fig 5: Pyridostatin is neurotoxic for primary cortical neurons(A) G-quadruplex is a non-canonical DNA secondary structure. Four guanine molecules (a single guanine is in red) can assemble into a square planar structure. The structure of a G-quadruplex is stabilized by hydrogen bonds between guanines and the interactions with a monovalent cation (Na+ or K+) resided in the central channel. (B) Repetitive guanine-rich DNA or RNA sequences have the potential to form G-quadruplex structures (C). (D) An example of survival analysis. Primary cortical neurons were transfected with mApple (a morphology and viability marker) and tracked with an automated microscope. Images collected every 24 h demonstrate the ability to return to the same field of neurons and to follow them over time. Each image is a montage of non-overlapping images captured in one well of a 24-well plate. Scale bar is 100 µm. A region from the original images at different time points is zoomed in to demonstrate longitudinal single-cell tracking (bottom panel). Red arrows depict two neurons that degenerate before 96 h after transfection. Note that neurites of the neuron 1 retract overtime. Scale bar is 20 µm. (E) Primary cortical neurons were transfected with mApple and treated with a vehicle or with different concentrations of pyridostatin (PDS; 0.01–5 µM). Transfected neurons were tracked with an automated microscope. Risk of death curves demonstrate that pyridostatin is neurotoxic. ***p<0.0001 (log-rank test). N.s., non-significant. Three hundred neurons were analyzed from three independent experiments. (F) Primary cortical neurons were treated with a vehicle (upper panel, control) or with 1 µM pyridostatin overnight (lower panel; PDS), fixed and stained with antibodies against MAP2c (red) and synapsin (green). Scale bar is 10 µm. (G) Quantification of the synapsin fluorescence intensity from (F). ***p<0.0001 (t-test). (H) Quantification of the neurite density from (F). MAP2c staining was used by the algorithm to identify and analyze neurites. ***p<0.001 (t-test). Three hundred neurons were analyzed from two independent experiments.