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Figure 3

Figure 3. Overexpression of Rab7N161T inhibits axonal transport of TrkA-mCherry, while a dominant negative Rab7 mutant (Rab7N125I) has no apparent effect on axonal transport of TrkA-mCherry in DRG neurons. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

(A) Kymographs of EGFP-Rab7N161T and TrkA-mCherry signals in co-transfected axons showing that signals that contained TrkA-mCherry only moved faster than endosomes containing both EGFP-Rab7N161T and TrkA-mCherry. (B) The average transport speeds of EGFP-Rab7N161T only, TrkA-mCherry only, or co-transfected with EGFP-Rab7N161T/TrkA-mCherry in DRG neurons were quantitated and shown. (C) A representative axon co-transfected with the dominant negative EGFP-Rab7N125I mutant and TrkA-mCherry as analyzed by kymograph. Transport of TrkA-mCherry was not affected by the expression of the dominant-negative EGFP-Rab7N125I.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 2

Figure 2. CMT2B Rab7 mutants move faster than Rab7wt within DRG axons, but slower than TrkA-mCherry. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

(A) Average transport speeds of singly transfected TrkA-mCherry and various EGFP-Rab7 constructs (wt, Q67L, L129F, K157N, N161T, V162M, N125I, and T22N). The different Rab7 constructs (Q67L, L129F, K157N, N161T, and V162M) moved significantly faster than Rab7wt, but much slower than TrkA-mCherry at room temperature. Axonal transport of the N125I and the T22N mutants were not registered since neither showed punctuated patterns. Similarly, at 37°C, the Q67L and N161T mutants moved significantly faster than the wt (inset). (B) A detailed comparison of retrograde and anterograde axonal transport speed between EGFP-Rab7wt, EGFP-Rab7Q67L, and EGFP-Rab7N161T for the data set in the inset of A. EGFP-Rab7wt, EGFP-Rab7Q67L, and EGFP-Rab7N161T moved at similar speeds in the retrograde direction. However, both EGFP-Rab7Q67L and EGFP-Rab7N161T traveled much faster than EGFP-Rab7wt in the anterograde direction. (C) Percentile of endosomes transported in both directions. Percentage of anterograde endosomes increases for EGFP-Rab7N161T compared to EGFP-Rab7wt and EGFP-Rab7Q67L. (D) Histograms of retrograde versus anterograde speed for EGFP-Rab7wt, Rab7Q67L, and EGFP-Rab7N161T. A fast anterograde transporting population (2.0–4.0 μm/s) appears for both EGFP-Rab7Q67L and EGFP-Rab7N161T compared to EGFP-Rab7wt.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 8

Figure 8. A proposed model for the pathogenic mechanism of CMT2B Rab7 mutations. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

NGF binds to and activates TrkA at the surface leading to activation of the PI3K/Akt pathways. However, activation of the Erk1/2 signaling cascade requires endocytosis of the NGF/TrkA signaling complex into Rab5-early endosomes, i.e. signaling endosomes. The signaling endosomes transmit the NGF/TrkA/pErk1/2 trophic signal to the soma via retrograde axonal transport. Under normal circumstances, upon completion of the delivery, the trophic signaling endosomes are down-regulated following fusion with the Rab7-late endosomes/lysosomes/autophagosomes (A). However, in CMT2B, the mutations cause acceleration in anterograde axonal transport of Rab7-late endosomes/lysosomes/autophagosomes, which likely leads to hyper-activation of these degradation organelles within axons. Endocytosed NGF/TrkA signals encounter and fuse with these Rab7-late endosomes/lysosomes/autophagosomes prematurely. The low pH value and the abundant presence of degrading proteases within Rab7-vesicles will result in premature termination of the NGF/TrkA signal, e.g. Erk1/2. As a result, retrograde transmission of trophic signals is disrupted leading to axonal degeneration and neuronal atrophy (B).

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 5

Figure 5. RNAi-mediated knockdown of Rab7 causes abnormal accumulation of TrkA-mCherry signals within large Rab5-endocytic vesicles. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

PC12M cells were cultured and transfected with TrkA-mCherry alone, or together with the GL3 control siRNA, Rab7 siRNA-1, or Rab7 siRNA-2. Non-transfected cells as well as transfected cells were harvested for Western blotting analysis using specific antibodies as indicated (A). Both Rab7 SiRNA-1 and -2 specifically reduced Rab7 expression efficiently. The levels of Rab5A, Rab5B, and Rab11A were not affected. GAPDH was blotted as well and serves as a loading control. (B) Representative images are shown for cells expressing TrkA-mCherry alone (top left), TrkA-mCherry+GL3 siRNA (top right), TrkA-mCherry+Rab7 siRNA-1 (bottom left), and TrkA-mCherry+Rab7 siRNA-2 (bottom right). The size distribution of TrkA-mCherry endosomes in each conditions (n=5) was quantified using ImageJ. Both Rab7 SiRNA-1 and -2 induced enlarged TrkA vesicles (C). In (D), cells were cultured on glass coverslips and co-tranfected with Rab7 siRNA-1, EGFP-Rab5wt, and TrkA-mCherry. Live cell imaging was performed to determine colocalization of EGFP-Rab5wt and TrkA-mCherry. A representative cell is shown. Colocalization of TrkA-mCherry with EGFP-Rab5wt is indicated with arrowheads.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 6

Figure 6. CMT2B Rab7 mutants show enhanced activation (i.e. GTP-bound) and markedly suppress the NGF-Induced Erk1/2 activation. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

As in Figure 5, PC12M cells were cultured and transfected with indicated constructs. Control and transfected cells were treated with 50 ng/ml NGF for 30 min and cell lysates were harvested for immunoblotting. In (A), the lysates were analyzed using an antibody against GFP to show that all the EGFP-Rab7 constructs were expressed at about the same level. In (B), cell lysates were incubated with GTP-agarose beads to pulldown activated Rab7 proteins. The amounts of EGFP-Rab7 proteins were analyzed by SDS-PAGE and blotted with an antibody against GFP. The blot in (A) was then reprobed with an antibody against Rab7A to indicate the endogenous levels of Rab7A under all conditions (C). In (D), the lysates are probed with antibodies against pAkt, total Akt, and in (E) with antibodies against pErk1/2 and total Erk1/2. A non-treated sample was also included as a control. For (D) and (E), representative blots within linear exposure were quantitated using ImageLab (Bio-Rad) and the results are shown. The level of activated pAkt (D) and pErk1/2 (E) in response to NGF treatment in untransfected cells was set at 100%.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 7

Figure 7. Expression of CMT2B Rab7 Mutants inhibits NGF-induced differentiation in PC12M cells and causes axonal degeneration in DRG neurons. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

In (A and B), PC12M cells were transfected, treated with 50 ng/ml NGF for 3 days, and then the length of neurites was measured and quantitated. Representative images are shown in (A) and the quantitation is shown in (B). Similar to EGFP-Rab7Q67L, expression of EGFP-Rab7L129L and EGFP-Rab7V162M resulted in significant inhibition of NGF-induced differentiation in these cells. In (C), the TrkA-GFP construct was cotransfected into PC12M cells with either Rab7 siRNA-1 (bottom panels) or GL3 control siRNA (top panels). Cells were then treated with NGF (50 ng/ml) for 3 days to induce differentiation. Cells were fixed and stained with an antibody against tubulin and DAPI to stain for the nuclei. The GFP images are shown in the left panels, and the images of tubulin and DAPI for revealing the cell morphology are shown in the right panels. Knockdown of Rab7 did not affect cell differentiation. In (D), rat E15/16 DRG neurons were cultured for three days and then transfected using Magnefect-NanoII (Nanotherics), a technique that allowed us to transfect adhered neurons and preserve the existing axons/neurites. After 72 hours, the length of the axons was measured using ImageJ and the quantitative results are shown. Expression of EGFP-Rab7V162M causes shortening of axons.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 4

Figure 4. Overexpression of activated Rab7 constructs, but not the dominant negative Rab7T22N mutant, inhibits NGF binding and internalization and reduces surface TrkA. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

PC12M cells were transfected with EGFP-Rab7wt (A) EGFP-Rab7L129F (B) EGFP-Rab7N161T (C) and EGFP-Rab7Q67L (D) using Lipofectamine 2000 as before. Cells were pre-treated with 0.2 nM Qdot-NGF for 30 min on ice, washed, and warmed up to 37°C for 20 min. Live cell imaging was carried out to detect EGFP-Rab7 constructs (middle panels in A, B, and C) and Qdot-NGF signals (bottom panels in A, B, and C). Expression of EGFP-Rab7wt, EGFP-Rab7L129F, and EGFP-Rab7N161T resulted in a marked reduction of Qdot-NGF signals from either the membrane or the cytoplasm (white-dashed profile). In non-transfected cells (yellow-dashed profile), Qdot-NGF was readily observed. The DIC images are presented in the top panels (A, B, and C). Similar results were seen in EGFP-Rab7Q67L–expressing cells (D). In contrast, expression of EGFP-Rab7T22N did not affect Qdot-NGF binding and internalization (E). Cells that expressed EGFP-Rab7L129F (F) or EGFP-Rab7V162M (G) were fixed and stained for surface TrkA with the rabbit RTA IgGs, followed by nuclear staining. In comparison to untransfected cells, cells that expressed either EGFP-Rab7L129F (F) or EGFP-Rab7V162M (G), as indicated with arrowheads, showed a marked reduction in RTA signals. Representative images are shown.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.
Figure 1

Figure 1. Expression profiles of CMT2B Rab7 mutants in PC12M cells and in rat DRG neurons. From: Defective Axonal Transport of Rab7 GTPase Results in Dysregulated Trophic Signaling.

(A) Fluorescent images of EGFP labeled wildtype (wt), constitutively active (Q67L), dominant negative (N125I and T22N), and the four CMT2B Rab7 mutants (L129F, K157N, N161T, and V162M) expressed in PC12M cell lines. Rab7wt accumulates as small puncta structures, whereas the constitutively active and CMT2B mutants generate ring-shape vesicular structures. The dominant negative Rab7 mutants are diffused within the cytoplasm. (B) Cells that expressed EGFP-Rab7V162 were fixed and stained for lysosomes with rabbit anti-LAMP3 IgGs or for autophagosomes with rabbit anti-p62 IgGs. Nuclear staining was also performed. Representative images are shown (a: LAMP3; b: p62). Colocalization is marked with arrowheads. (C) Expression of the wildtype, the constitutively active (Q67L), the CMT2B mutant N161T, and TrkA-mCherry in DRG neurons visualized by fluorescent microscopy with corresponding DIC images shown. (D) Snapshots of endosomes containing TrkA-mCherry or EGFP-Rab7 in DRG axons. TrkA-mCherry signals show a puncta pattern in DRG axons (top left panel), and a similar pattern is seen for EGFP-Rab7Q67L and EGFP-Rab7N161T (top middle and right panels). The puncta pattern for EGFP-Rab7wt is less pronounced than EGFP-Rab7Q67L and EGFP-Rab7N161T (bottom left panel). The dominant negative form, EGFP-Rab7T22N shows a diffused pattern (bottom middle panel). A micro-channel in a microfluidic device is indicated to determine the direction of axonal transport (bottom middle panel). A merged image between the EGFP-Rab7N161T channel and its DIC channel is also presented to indicate typical transfection efficiency (bottom right panel). (E) Representative kymographs of axonal transport of endosomes containing EGFP-Rab7Q67L are shown with the transport speeds of two segments computed.

Kai Zhang, et al. J Neurosci. ;33(17):7451-7462.

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