Importance of the Rab3a-GTP Binding Domain for the Intracellular Stability and Function of Rabphilin3a in Secretion

We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51 190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn 2+ -binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66356/1/j.1471-4159.1997.69010164.x.pd

CNTF Induces Regeneration of Cone Outer Segments in a Rat Model of Retinal Degeneration

Cone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter.Recombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function.Loss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations

CNTF induces regeneration of cone outer segments in a rat model of retinal degeneration.

BackgroundCone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter.Methodology/principal findingsRecombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function.Conclusions/significanceLoss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations

Photoreceptors in Sprague-Dawley rats.

<p>Plastic-embedded sections of retinas from wild-type Sprague-Dawley rats of PD 8 (A), 12 (B), 16 (C), 20 (D), 30 (E), and 60 (F) were examined by light microscopy (superior regions). The ONL is indicated in each panel by a white vertical bar. No progressive loss of photoreceptors is observed. Sections were stained with toluidine blue. Scale bar, 20 µm.</p

Specificity of PNA staining to COS.

<p>Whole-mounted retinas (superior regions) of S334ter-3 rats (PD 30) were double stained with PNA and antibodies against red/green opsin (A) or blue opsin (B). PNA-staining overlaps with red/green opsin or blue opsin staining very well. The specificity of PNA staining to COS was confirmed under high magnification (C). PNA stained almost exclusively COS (C), whereas antibodies against blue opsin stained not only COS, but also, to a lesser degree, the inner segment, the cell body, and the synaptic terminal (C). Scale bar: 50 µm for A and B; 10 µm for C.</p

Regeneration of COS induced by CNTF treatment.

<p>Retinas (superior regions) of S334ter-3 rats at PD 35 were treated with either CNTF or PBS and harvested 10 days after treatment. Whole-mounted retinas of PD 35 (A, before treatment), PD 45 treated with PBS at PD 35 (B), and PD 45 treated with CNTF at PD 35 (C) were stained with PNA. PNA positive cells in CNTF treated retinas are more than those in either PBS treated retinas or PD 35 retinas. Quantitative analysis showed that PNA-positive cells are significantly more in the PD 45 CNTF treated retinas (657±76, n = 4) than in the PD 35 retinas (467±73, n = 3), or PD 45 retinas treated with PBS (334±33, n = 3) (D, mean ± SD), indicating regeneration of COS in response to CNTF treatment. Asterisk indicates <i>P</i><0.05; double-asterisk indicates <i>P</i>≤0.01 (ANOVA analysis and Bonferroni test). Scale bar: 100 µm.</p

Long-term effects of sustained delivery of CNTF.

<p>Eyes of S334ter-3 rats were implanted with microdevices containing CNTF-secreting cells or parental cells at PD 20 and harvested on PD 160. Whole-mounted retinas (superior regions) were stained with PNA or antibodies against red/green opsin. PNA or red/green opsin positive cells are more in the retinas treated with CNTF devices (A and B than the fellow control retinas (C and D). Quantitative analysis of PNA or red/green positive cells in the superior retina showed significantly more cells in CNTF-treated retinas (446±80 and 310±46, respectively) than the controls (141±40 and 108±46, respectively) (E, mean ± SD, n = 3). Cone ERGs were recorded at PD 135 from eyes implanted at PD 20 with CNTF secreting or control devices. Representative b-waves from CNTF treated and control eyes are shown in panel F. The onset of flash is indicated by an arrowhead. The amplitudes of b-waves from CNTF treated retinas (32.46±10.02 µV) is significantly larger than from control retinas (17.70±4.78 µV) (G, mean ± SD, n = 3). Asterisk indicates <i>P</i><0.05; double-asterisk indicates <i>P</i><0.01 (Student <i>t</i>-test). Scale bar: 50 µm.</p

Increase in PNA-positive cells after CNTF treatment.

<p>Retinas (superior regions) of PD 10, 20, 50, 80, and 170 S334ter-3 rats were treated with either CNTF (left eyes) or PBS (right eyes) and collected 10 days after treatment. Whole-mounted retinas of PD 20 (i.e., injected at PD 10; A, CNTF; D, PBS), PD 30 (B, CNTF; E, PBS), PD 60 (C, CNTF; F, PBS), PD 90 (G, CNTF; I, PBS), and PD 180 (H, CNTF; J, PBS), were stained with PNA. Many PNA-negative areas appeared in PBS treated retinas, whereas no PNA-negative areas were seen in the PD 20 retinas treated with CNTF (A). The PNA-negative areas became much smaller and in most cases completely disappeared in PD 30 and 60 retinas treated with CNTF (B and C). Increase in PNA positive cells after CNTF treatment became less dramatic in PD 60 retinas (G), and by PD 180 (H), little or no significant increase is observed. Scale bar: 200 µm.</p

Loss of COS in the retinas of S334ter-3 rats.

<p>Whole-mounted retinas (superior regions) of S334ter-3 rats (Tg) are shown at PD 10 (A), 20 (B), 30 (C), 60 (D), 90 (E), and 180 (F), as well as retinas of wild-type (Wt) rats at PD10 (G), 20 (H), and 60 (I). Retinas were stained with PNA and examined by confocoal microscopy. In S334ter-3 retinas, loss of PNA staining is concentrated in small round or irregularly shaped areas. The loss of PNA staining was progressive with age (A–F). Quantitative analysis of the density of PNA positive cells in the S334-ter-3 retinas clearly shows the progressive loss of PNA positive cells in the superior retina (J, mean ± SD, n = 4). No such loss of PNA positive cells was found in wild-type retinas (G–I). Scale bar: 200 µm.</p