EMBO/soda-nel huggingface dataset:
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf2

(a) qRT-PCR analysis for Opn1sw during cone degeneration. (b–o) All panels show retinal flat mounts except for i and l. Green signal indicates PNA expression and red signal indicates red/green or blue opsin. The wild-type retina is shown at P35 in b–d. Red/green opsin (b) and PNA (c, d) expression were detected both dorsally and ventrally, whereas blue opsin (c,d) was detected only ventrally. The Pde6b−/− mouse is analyzed in e–g and j–o. A central to peripheral gradient of PNA and shortening of cone outer segments is shown in e–g. At P20, there were fewer elongated outer segments in the center (e) as compared with the periphery. Higher magnifications of a central or peripheral outer segment from e are shown in f and g, and a wild-type outer segment is shown in h (white line marks the outer segment). A quantification of outer segment length at 3 weeks is shown in i (error bars represent s.d. of 15 measurements each). With the shortening of outer segments during degeneration, red/green opsin was localized throughout the membrane of the cell body, and PNA, which detects an extracellular protein(s), was reduced to a small dot attached to the residual outer segment (j) (yellow shows red/green and PNA overlap, arrow). A higher magnification of a cone showing red/green at the membrane is displayed in k (arrow). A cross section showing red/green in cell body is shown in l (arrows) (P70; j–l). During degeneration, red/ green opsin was detected mainly dorsally (m), whereas PNA (m, n) or blue opsin (o) expression were not altered (m and n, P21; o, P49). GCL, ganglion cell layer; INL, inner nuclear layer.
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf3

(a) Schematic representation of the rod and cone death kinetics found in the four mouse models of retinitis pigmentosa. The onset of cone death is set as time zero. The corresponding time windows on the x axis are given in weeks. The major cone death phase was the time period from onset until roughly 85% of cones had died. The end phase of cone death was the time period thereafter. (b) Summary of rod and cone death kinetics. Time is indicated in postnatal days or weeks. (c) Immunofluorescence on retinal flat mounts showing the ventral reduction of red/green opsin expression found in the four mutants during cone degeneration. Strain and time, in postnatal days, are indicated below each image (for higher magnification of wild type, see Fig. 2b).
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf5

(a–c) P-mTOR levels in wild-type retinae. Dorsal (top) enrichment of P-mTOR (a). Higher magnifications of the dorsal and ventral regions are shown to the right, with P-mTOR in red and cone segments in green, as detected by PNA. Dorsal retinal sections stained for P-mTOR (red) and PNA (b) or antibody to β-galactosidase (green, c). Higher magnification (insets) images suggest that the P-mTOR signal is located in the lower part of the outer segment (OS). IS, inner segment. (d–g) Rapamycin treatment of wild-type mice led to downregulation of red/green opsin ventrally (e) but not dorsally (red, d). Ventral blue opsin (red, f) remained unaffected, as did PNA (d–g) (green) and P-mTOR itself (red, g). (h–m) Reduced levels of dorsal P-mTOR during photoreceptor degeneration (red). The wild-type control is shown in h and the Pde6b mutant is shown in i and j. Reduction started during rod death at P15 (i) as the outer segments (green, PNA) detached from the retinal pigmented epithelium. By P30, only a few cones (green signal: α-β-galactosidase) showed high levels of P-mTOR (red) (j). (k–m) Similar reduction was seen in dorsal cones of the other three mutants (cones marked in green by PNA; Pde6g−/− is shown at P35 in k; Rho−/− is shown at PW20 in l; P23H is shown at PW70 in m). All panels show immunofluorescence on retinal flat mounts (photoreceptor side up) with the exception of b, c and g, which show retinal sections. Blue indicates DAPI.
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf6

All panels show immunofluorescent staining. Blue shows nuclear DAPI staining and green shows cones marked with PNA. (a–f) Staining for HIF-1α (red signal). (a) Wild type (PW10) stained for HIF-1α (inset shows higher magnification). (b, c) Cross sections in wild type stained for HIF-1α (PW10). DAPI overlap is shown in c. (d–f) During cone degeneration in Pde6b−/− (PW10) mice, we found increased levels of HIF-1α in cones (inset in d shows higher magnification with DAPI overlap). Cross sections indicated that the increase of Hif-1α occurred mainly in cones (arrows point to cones that are located in the top layer of the inner nuclear layer at this stage; e). DAPI overlap is shown in f. (g) GLUT1 expression in wild type (PW10) (red). Most of the signal in between the cones reflects expression in rods. (h–j) Increased expression of GLUT1 in cones during degeneration seen in flat mounts (h) and sections (i, j). PNA overlap of j is shown in i. Retinal flat mounts are shown in a, d, g and h. Retinal sections are shown in b, c, e, f, i and j.
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf7

(a–c) Immunofluorescence on retinal flat mounts (LAMP-2 is shown in green, red/green opsin in red and DAPI in blue). Insets show enlarged cells (arrow). A wild-type retinae at PW5 showing a lysosome (small green dots) with normal LAMP-2 distribution is imaged in a. Weak red/green opsin signal was detected at the level of the photoreceptor nuclei, as it was mainly found in the outer segments in the wild type. Enlarged lysosomes (dots) resulting from an accumulation of LAMP-2 at the lysosomal membrane were seen specifically in cones from Pde6b−/− mice PW5 (b). Confocal section of same field as in b taken at the level of the inner nuclear layer showed LAMP-2 levels similar to those in wild type (c). (d) qRT-PCR for the three different LAMP-2 splice forms showing the relative concentration and the ratios between the Pde6b−/− mutant and wild type. Error bars represent s.d. of three measurements.
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf8

(a–c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig. 9). An example of untreated control is shown in a, an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c. (d) Quantification of cone survival after four weeks of treatment. Data represents an average of at least eight retinae. The y axis represents the percentage of the cone surface area versus the surface area of entire retina (see Supplementary Figs. 8 and 10). (e, f) Measurements of blood glucose levels (e) and body weight (f) performed weekly over the time span of the experiment. (g, h) Immunofluorescent staining on retinal flat mounts for HIF-1α (red) and PNA (green) in untreated control Pde6b−/− (g) and Pde6b−/− mice treated for 4 weeks with insulin (h). DAPI is shown in blue. Error bars in d–f represent s.d.
Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons (2022)
Figure 1

Figure 1 Injury-induced Rpt3 CKO mice decrease the mitochondrial number in injured motor axons.

A Schematic diagram of hypoglossal nerve injury model. The axons of motor neurons in hypoglossal nucleus (XII) are projected to tongue muscle. Unilateral hypoglossal nerve is transected. Red box indicates the peripheral part of injured nerve observed in (F) and (G).

B Schematic diagram for injury-induced deletion of proteasome subunit, Rpt3, from motor neurons. Atf3:BAC2 transgene and floxed Rpt3 gene are presented. The initiation codon of Atf3 in BAC is replaced by that of Mito-AcGFP. Black and white boxes indicate coding and non-coding regions of exon respectively. Arrowheads denote loxP sites. Rpt3 deletion and mitochondrial labeling occur at the same time in injured motor neurons. See Fig EV1 D-G and Materials and Methods for details.

C The immunostaining of GFP and RPT3 protein in hypoglossal nucleus (XII) of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days (5d) after axotomy. Arrows show Rpt3-deleted injured motor neurons. cc, central canal.

D 3D images of GFP-labeled mitochondria (mtGFP) in the transparent brainstem of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after hypoglossal nerve injury. Inset shows the direction and portion observed by light sheet microscopy. White arrows indicate the magnified area shown in (E). D, C and L indicate dorsal, caudal and lateral directions respectively.

E The magnified injured axons labeled by mtGFP.

F Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury.

G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury.

H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group).

I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group).

J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). See also Fig EV2G and H.

Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in (C), midline of the brain in (D) and axon in (F and G). Scale bars, 150 μm in (C, top) and 20 μm in (C, middle and bottom), 500 μm in (D), 50 μm in (E), 5 μm in (F) and 2.5 μm in (G).
Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons (2022)
Figure 3

Figure 3 The injury-induced ablation of Rpt3 affects the disassembly of the AIS in injured motor neuron.

A Schematic diagram of the AIS in hypoglossal nerve injury model.

B Schematic showing the time course of motor neurons after nerve injury. Arrows indicate the time point at which the experiments were performed.

C Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm.

D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.

E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.
Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons (2022)
Figure 4

Figure 4 The proteasome is responsible for the AIS-specific degradation of AnkG in injured motor neurons

A Graph represents fold change of mRNAs in injured hypoglossal (XII) nuclei compared with control hypoglossal nuclei of wild type mice at 5 days after injury. Data are shown as the mean ± s.e.m. (n = 3 independent experiments).

B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.

C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.

D The expression of AnkG (red) and GFP (green) in the AIS of hypoglossal (XII) motor neurons and that of AnkG, GFP and Caspr (blue) in nodes of hypoglossal motor nerves at 5 days after injury. Scale bars, 15 μm, 5 μm and 1.5 μm from the top.
Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons (2022)
Figure 5

Figure 5 The absence of the AIS facilitates mitochondrial influx to injured axons.

A Schematic diagram of hypoglossal nerve injury model. The areas shown by the red boxes (the AIS and hypoglossal (XII) nerve) are observed in this figure.

B Time-dependent localization of AnkG and GFP-labeled mitochondria in motor neuron (MN) of Atf3:BAC2 Tg mouse before and after hypoglossal nerve injury.

C The localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice.

D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon.

E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group).

F Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon.

G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice).

H Schematic diagram demonstrating the AAV infection to tongue and the AAV constructs.

I Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury.

J Fluorescent intensity scans of GFP and AnkG corresponding to (I).

K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group).

L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal.

M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group).

Data information: For (E, G, K and M), data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E and G), ** P = 0.0064 in (K), * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In (B, C and I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in (B, C, F and I) and 100 μm in (L).
Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons (2022)
Figure 7

Figure 7 Failure of the AIS disassembly and mitochondrial influx in stress-responsive GFP motor neurons of Atf3;SOD1 mouse.

A Breeding schemes to obtain Atf3:BAC; SOD1G93A (Atf3;SOD1) mouse.

B 3D images of GFP-labeled mitochondria (mtGFP) in the transparent spinal cord without injury of Atf3;SOD1 mouse at pre-symptomatic stage (P70, 70-day-old) and Atf3:BAC Tg mouse (inset). V, C and L indicate ventral, caudal and lateral directions respectively.

C Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord.

D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group).

E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group).

F Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70.

G Quantification of the AIS length (n = 5 mice per group).

H The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS.

I Fluorescent intensity scan of GFP and AnkG in corresponding to (H).

J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group).

K Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon.

L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice)

Data information: For (D, E, G, J and L), data are shown as the mean ± s.e.m., no significant difference in (G), ** P = 0.0008, *** P = 0.0007 in (J), ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 700 μm in (B), 1000 μm in (B; inset), 50 μm in (C; upper panel), 20 μm in (C; lower), 10 μm in (F and K) and 5 μm in (H). See also Fig. EV5.
Who with whom: Functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation (2020)
Figure 2

Figure 2. Hrd1 selects Ubc7 through high binding affinity.

A Dissociation constants for ERAD E2-SS-Ub/E3 pairs. KD values were determined by ITC titration of Hrd1 or Doa10 to Ubc6-SS-Ub and U7BR/Ubc7-SS-Ub, respectively. Due to the weak binding, errors are high, so the constants are reported to just a single significant figure.

B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reactions do not contain S-Hrd1, "no ATP" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown.

C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top - yellow) and Ubc6 (bottom - green) with ethanolamine as nucleophile. Representative Coomassie gels are shown.

D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05.

(E) Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by Hrd1. E2 stimulation is reported as the ratio of rates derived from RING-catalyzed reactions and "no E3" controls in D. Values are reported as means ± standard deviation (n = 3). Significance was determined by student T-test; * p < 0.05.
Who with whom: Functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation (2020)
Figure 3

Figure 3. Ubc7-mediated Ub chain elongation is not rate-determining for protein degradation.

A In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2C-D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set.

B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown.

C Steps of ERAD protein degradation with associated rate constants; S = ER protein; S* = misfolded protein, i.e. ERAD substrate; kobs = observed rate of protein degradation in assays; kUb1 = rate of the priming reaction; kUb2 = rate of the first elongation step, i.e. mono-Ub to di-Ub; kUb3 - kUbn = rates of subsequent elongation steps; k0 - kj = rates for other steps of ERAD; the black box identifies steps of substrate poly-ubiquitylation with priming highlighted by the green box and chain elongation by the yellow box. The light yellow background highlights the mono-Ub to di-Ub reactions.

D Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2).