Article Figures & Data
Figures
- Figure 1.
Cytoplasmic versus nuclear localization of hnRNP K shifted in RGCs at the peak period of axon regrowth. A1–D3, Retinal sections were immunostained for hnRNP K (green) and the cytoplasm was counterstained by immunostaining for S6, a ribosomal protein (red), at early [3 d after crush (A1–B3)] and peak [11 d after crush (C1–D3)] phases of axonal regrowth to the optic tectum. CLSM images (63× objective) were taken from the operated and contralateral unoperated eyes within the same section. At 3 d after crush, immunostaining was comparable between the operated and unoperated eyes, with hnRNP K immunoreactivity readily detectable in the cytoplasm as well as in the nucleus of RGCs (A1–B3, arrowheads). At 11 d, hnRNP K immunostaining in RGCs of the operated eye was sharply reduced in the cytoplasm and intensified in the nucleus; nuclear staining surrounded a relatively poorly stained nucleolus, positively stained for S6 (D1–D3, arrows indicate examples of cytoplasmic areas exhibiting significantly reduced hnRNP K staining). In contrast, hnRNP K staining in RGCs of the unoperated eye at 11 d resembled those of both the operated and unoperated eyes at 3 d (C1–C3, arrowheads indicate examples of cytoplasmic staining for hnRNP K). Scale bar in A1 applies to all panels.
- Figure 2.
Quantification of nuclear versus cytoplasmic localization of hnRNP K during regeneration. A1–B3, Representative sections of retina (11 d after crush) immunostained for hnRNP K (green) and nuclear counterstained with 7-AAD (red) from the operated (B1–B3) and contralateral unoperated (A1–A3) eyes within the same section. Circles (A3, B3) indicate examples of the cytoplasmic (between the inner and outer circles) and nuclear (inner circle) regions analyzed for an individual RGC. A1, A3, Arrowheads indicate examples in the unoperated eye of RGCs having cytoplasmic immunostaining. A3, Scale bar applies to all panels. C, Ratio of the area-normalized average intensity of hnRNP K IF in the nucleus versus the cytoplasm of RGCs (mean ± SEM) of the OEs versus contralateral UEs of the same sections. Nuclear versus cytoplasmic staining was significantly greater (p < 0.001, t test) in OE (10.4 ± 0.6) versus UE (1.9 ± 0.1) at 11 d, but not at 3 d (OE, 1.7 ± 0.1; UE, 1.8 ± 0.1; N.S., not significant; p = 0.4, t test, n = 3 animals). The ratio in the UE at 11 d was also not significantly different from that at 3 d (p = 0.16). D, Fluorescence intensities were not significantly different between RGCs of the OE versus those of the contralateral UE at either time point [OE/UE (±SEM): 3 d, 96 ± 7%, p = 0.5; 11 d, 103 ± 3%, p = 0.7; t test, n = 3 animals].
- Figure 3.
Antisense VMO suppressed hnRNP K expression in RGCs. Following repeated, unilateral, intravitreal injection of VMO, unoperated frogs were killed at the indicated time points, processed for immunofluorescence, and viewed by CLSM. A1–C2, Severe depletion of hnRNP K expression from RGCs, but not INs in eyes injected with hnRNP K VMO (lower micrograph, VMO inj.) compared to the contralateral uninjected eye (upper micrograph, Uninj.). D1, D2, Nuclear counterstaining (7-AAD) of retinal sections depicted in C1 and C2. Arrowheads indicate examples of RGCs in which hnRNP K protein expression was undetectable by immunostaining (C2, Arrowheads with dotted lines circumscribing the position of cell). E, F, Quantification of the ratio of hnRNP K immunofluorescence intensity per cell between the hnRNP K VMO-injected (PK VMO inj.) and uninjected eyes (mean ± SEM%) confirmed that suppression of hnRNP K expression was significant at all time points for RGCs (E, p < 0.001, t test, n = 4 animals) but not for interneurons (F, p = 0.3, 0.2, and 0.2, for 1, 3, and 12 d, respectively, t test). H, Density (average number of 7-AAD+ RGCs/100 μm of retinal arc per animal ± SD) of 7-AAD-labeled nuclei in the RGC layer was not significantly different between hnRNP K VMO-injected (VMO inj.) and uninjected (Uninj.) eyes at 12 d (p = 0.29, t test). I1–K2, Immunofluorescence indicated that hnRNP K VMO (12 d) affected neither the expression of hnRNP E2 (I1, I2) and peripherin (K1, K2) in retina nor of tau (J1, J2) in optic nerve. L1, L2, Mismatched control VMO (12 d) had no effect on hnRNP K immunofluorescence in RGCs. G, Quantification of data illustrated in I1, I2, L1, and L2 (Inj. eye/Uninj. eye; mean ± SEM; p = 0.2 for I1 and I2; p = 0.25 for L1 and L2; t test, n = 3 animals). Scale bars, 50 μm. Scales bar in A1 also applies to A2–B2; scale bar in C1, also applies to C2–D2; scale bar in I2, also applies to I1; scale bar in J2 also applies to J1; scale bar in K2 also applies to K1; scale bar in L2 also applies to L1.
- Figure 4.
hnRNP K knockdown inhibited optic axon regeneration. B1–I2, Animals were subjected to orbital optic nerve crush on both sides but injected with hnRNP K VMO (B2, C2, D2, E2, F2, G2), mismatched control VMO (H2), or saline (I2) on only one side. Animals were processed for immunostaining at 12 d after crush. A1–A3, Tracings of representative transverse sections of the optic nerve at progressively more caudal levels from where the optic nerve exits the eye (A1) to the optic chiasm (A3). Approximately 36 sections are between A1 and A2. Approximately 52 sections are between A2 and A3. B1–I2, Images of the optic nerve were taken from either near the lesion site (B1–C2, at the position illustrated in A1) or near the position illustrated in A2 (D1–I2). All images of the injected and uninjected sides were taken from the same sections. B1, C1, On the uninjected side, N-β-tubulin-positive regenerating optic axons penetrated the lesion site (B1, LS) to occupy the periphery of the optic nerve in the immediately adjacent section (C1, arrow). B2, C2, On the hnRNP K VMO-injected side, axons stopped at the lesion site (B2, LS) and could not be detected in the section immediately beyond [C2, ON (optic nerve); staining of the adjacent peripheral nerve (PN) indicates that the immunostaining procedure was successful]. D1–G2, Immunostaining of hnRNP K VMO-injected animals for axonal markers [N-β-tubulin (D1, D2), GAP-43 (E1, E2), NF-M (F1, F2) by immunofluorescence] or the intravitreally injected axonal tracer WGA (G1, G2; by immunoperoxidase). On the uninjected side, labeled regenerating axons circumscribed the core of degenerating axons (arrows) but were undetectable on the injected side (dotted ellipse indicates outer circumference of the optic nerve). H1–I2, N-β-tubulin staining of regenerating optic axons of control VMO-injected (H1, H2) or saline-injected (I1, I2) animals at 12 d after crush indicated that these treatments had no effect on regeneration. Scale bar in F1 also applies to D1–E2 and to F2; scale bar in G2 also applies to G1; scale bar in H2 also applies to H1; scale bar in I2 also applies to I1.
- Figure 5.
hnRNP K knockdown by itself did not induce neurodegeneration during optic axon regeneration. Frogs receiving bilateral, orbital optic nerve crushes and unilateral injections of hnRNP K VMO were killed at 12 d after crush. A1, A2, A3, Immunostaining for N-β-tubulin in the ganglion cell (GCL) and inner plexiform (IPL) layers was more robust at 12 d after crush (A2, A3) than in surgically naive, unoperated eye (A1) and in the hnRNP K VMO-injected eye (A3) than in the contralateral uninjected eye (A2). B1, B2, Uptake of WGA in RGCs was unchanged in the hnRNP K VMO-injected eye (B2), compared with the contralateral uninjected eye (B1). The density of WGA-labeled RGCs (RGCs/100 μm of retinal arc ± SD) was not significantly different between the uninjected and injected eyes (11.6 ± 0.3 vs 11.4 ± 0.5, respectively; p = 0.28, t test) (B2 became cropped in the lower right hand corner when the image was rotated). C1–C4, Fluoro-Jade C staining was similar between the retina of hnRNP K VMO-injected (C2) and uninjected (C1) eyes. In both eyes, Fluoro-Jade C staining of retina was much less intense than in cells adjacent to a spinal cord transection (C3). C4, Density of DAPI-labeled nuclei in the retinal ganglion cell layer (DAPI+ RGCs/100 μm of retinal arc ± SD) was not significantly different between hnRNP K VMO-injected (VMO inj.) and uninjected (Uninj.) eyes (N.S., p = 0.12, t test); although both exhibited a significant decrease (19% loss; *p < 0.01, t test on counts derived from 3 frogs) compared with the uninjured eye. D1, D2, Fluoro-Jade C staining of degenerating optic axons (dON) was similar between VMO-injected (D2) and uninjected (D1) eyes. D1, Dotted outline indicates the positions of regenerating axons, unstained by Fluoro-Jade C, surrounding the degenerating core. No such region is visible on the VMO-injected side. INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar in A3 applies to A1 and A2; scale bar in B2 applies to B1; scale bar in C2 applies to C1; scale bar in D2 applies to D1.
- Figure 6.
hnRNP K knockdown inhibited protein, but not mRNA, expression of select cytoskeletal-associated targets activated during optic nerve regeneration. A1–F3, Sections containing hnRNP K VMO-injected and contralateral uninjected eyes from animals receiving bilateral optic nerve crush 12 d earlier, and sections of uninjected eyes receiving no crush were processed for immunofluorescence (A1–A3, C1–C3, E1–E3; imaged with conventional epifluorescence) and digoxigenin-labeled in situ hybridization (B1–B3, D1–D3, F1–F3; imaged in brightfield) to compare protein with mRNA expression for NF-M (A1–B3), GAP-43 (C1–D3), and peripherin (E1–F3). mRNA expression of all three genes increased dramatically in the GCL with optic nerve crush, regardless of whether or not eyes were treated with hnRNP K VMO (B2, B3 vs B1; D2, D3 vs D1; F2, F3 vs F1). NF-M protein [mainly visible in RGC axons, in the nerve fiber layer (NFL)] and GAP-43 protein (mainly present in RGC dendrites, located in the IPL), increased during regeneration in only the uninjected eyes (A2, C2), whereas peripherin protein [which in unoperated eye is restricted to Mueller radial glia (E1)] increases in RGCs (E2, E3, arrowheads) in both hnRNP K VMO-injected and uninjected eyes. Scale bars, 50 μm. Scale bar in A3 applies to A1, A2; scale bar in C3 applies to C1, C2; scale bar in E3 applies to E1, E2; scale bar in B3 applies to B1, B2, D1–D3, F1–F3.
- Figure 7.
hnRNP K knockdown induces defects in the translation of select cytoskeletal-associated RNA targets during optic axon regeneration. A, Efficiency of nuclear export of NF-M, GAP-43, tau, and peripherin mRNAs. qRT-PCR of nuclear versus cytosolic fractions from eye was performed for each mRNA at 12 d after optic nerve crush under the indicated conditions (color key, right). ΔCt, difference in the number of PCR cycles to reach threshold (±SD) between the nuclear and cytosolic fractions (**p ≤ 0.01; *p ≤ 0.05, N.S., p > 0.25, t test on 3 replicates). B, Polysomal profiles of NF-M (top) and peripherin (bottom) RNA at 12 d after optic nerve crush under four different conditions, as indicated. P, polysomal fractions; SP, subpolysomal fractions; M, monosomal fractions; abscissa, fraction number; left ordinate and solid line, total RNA (A260); right ordinate and bars, percentage of NF-M or peripherin RNA loaded onto the gradient present in each fraction. C, The mean percentage (±SD) of each RNA in efficiently translated fractions (percentage RNA in polysomes) of NF-M, GAP-43, tau, and peripherin mRNAs under four different conditions (color key, right). In uninjected eyes, the efficiency of translation increased significantly for all four mRNAs during optic nerve regeneration. This regenerative increase was inhibited by hnRNP K VMO for the three hnRNP K mRNA targets (NF-M, tau, GAP-43) but not for the nontargeted mRNA (peripherin) (**p ≤ 0.01; N.S., p > 0.15; t test on 3 replicates).
