Brain Research 885 2000 53–61 www.elsevier.com locate bres
Research report
Early loss of synaptic protein PSD-95 from rod terminals of rhodopsin P347L transgenic porcine retina
a a,b
a a
a
Scott M. Blackmon , You-Wei Peng , Ying Hao , Suk Jin Moon , Leonardo B. Oliveira ,
a d
a,b,c ,
Misako Tatebayashi , Robert M. Petters , Fulton Wong
a
Department of Ophthalmology , Duke University School of Medicine, Durham, NC 27710, USA
b
Department of Neurobiology , Duke University School of Medicine, Durham, NC 27710, USA
c
Department of Pathology , Duke University School of Medicine, Durham, NC 27710, USA
d
Department of Animal Science , North Carolina State University, Raleigh, NC 27695, USA
Received 13 July 2000; accepted 29 August 2000
Abstract
Retinitis pigmentosa RP, a type of retinal degeneration involving first rod and then slow cone photoreceptor degeneration, can be caused by any of a number of mutations in different genes. In the cases of mutations affecting rod-specific genes such as rhodopsin, it is
unclear how the mutations may cause degeneration of cones. We have used the porcine retina, which is rod-dominated and has an abundance of cones, to study the mutation-induced changes in both rod and cone photoreceptors. Like patients with the same mutation,
rhodopsin P347L transgenic swine manifest rod–cone degeneration. In addition, the rod bipolar cells fail to form synaptic connections with rods; instead, they form ectopic synapses with cones. The mechanisms that prevent the formation of the rod–rod bipolar cell synaptic
connection are not known. We used specific antibodies and immunocytochemistry to show that the synaptic protein, PSD-95, is present in both normal and transgenic porcine retinas. During neonatal development, however, PSD-95 is lost from rod terminals in the transgenic
swine. This loss is virtually complete 90 by postnatal day 5, at a time when greater than 80 of rod cell bodies still remain. Furthermore, the remaining rods retain their outer segments and their gross morphology appears relatively normal. In contrast, PSD-95
expression continues in cone terminals, even in 10-month-old transgenic swine, where the rods have all disappeared and the cones show signs of severe degeneration. These results suggest that loss of PSD-95 may not be a general consequence of the deteriorating cell. Rather,
the very early and selective loss of PSD-95 from the rod terminals may be causally related to the absence of rod–rod bipolar cell synapses in the rhodopsin P347L transgenic retina.
2000 Elsevier Science B.V. All rights reserved.
Theme : Disorders of the nervous system
Topic : Genetic models
Keywords : PSD-95; Retinal degeneration; Retinitis pigmentosa
1. Introduction mutations affecting rod-specific genes such as rhodopsin,
however, it is unclear how the mutations may cause Retinitis pigmentosa RP is an inherited disorder in
degeneration of cones, which do not express rhodopsin. To which mutations in various retina-specific genes may lead
address this issue, we have created the rhodopsin P347L to rod and then slow cone photoreceptor degeneration
transgenic pig model [26], which has provided the oppor- [2,8,30]. In RP patients, the ultimate loss of cones impairs
tunity to study the mutation-induced changes in both rod visual function that is critical to their lives. Therefore, a
and cone photoreceptors. major challenge for RP research is elucidating the mecha-
Like patients with the same mutation, these transgenic nisms underlying cone degeneration. In the cases of
swine manifest rod–cone degeneration [26]. In these transgenic swine, in addition to photoreceptor death, the
rod bipolar cells fail to form synaptic connections with the
Corresponding author. Tel.: 11-919-684-8579; fax: 11-919-684-
rods; instead, they form ectopic synapses with and receive
8829. E-mail address
: fulton.wongduke.edu F. Wong.
input from the surviving cones [24,25]. This finding has
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 2 8 - 0
54 S
created a new conceptual framework to address the conse- serves as a rod-specific marker, and anti-G protein-gc
quences of the rhodopsin P347L mutation, such as discern- subunit polyclonal antibody CytoSignal, referred to
ing the mechanisms that prevent the formation of the hereafter as anti-cone transducin. G protein-gc is the g
rod–rod bipolar cell synaptic connection. subunit of cone transducin and thus serves as a cone-
Synapses are highly specialized structures, the formation specific marker [23].
of which requires the interactions of dozens of unique synaptic proteins. As a preliminary screen, we performed
2.3. Immunocytochemistry PCR to identify, in normal and transgenic porcine retinas,
some of the expressed genes that encode for proteins For immunoperoxidase cytochemistry, the retinal sec-
known to be associated with synapses [31]. Among the tions were incubated with mouse monoclonal anti-PSD-95
genes identified unpublished results of Blackmon and antibody 1:200 overnight at 48C, followed by three washes
Wong, PSD-95 is present in both normal and transgenic in PBS for 15 min each. Triton X-100 0.3 was added to
porcine retinas. This finding is consistent with recently all incubation and wash buffers. The sections were then
published results showing the presence of PSD-95 in the incubated with a biotin-conjugated anti-mouse secondary
mammalian retina, including the rat, monkey, and tree antibody 1:200, Vector Laboratories in PBS for 2 h at
shrew [17]. room temperature and then washed three times in PBS for
In the studies reported here, we have used specific 15 min each, followed by incubation with an avidin–
antibodies and immunocytochemical methods to determine biotin–peroxidase complex 1:100, Vector Laboratories in
the localization of PSD-95 in rod and cone terminals in the PBS for 1 h. After three more washes of 15 min each in
porcine retina. We discovered that the rod terminals of the PBS, the staining was developed with a substrate solution
transgenic swine lose PSD-95 during early postnatal life, of 20 ml of PBS, 0.1 ml of 3 hydrogen peroxide and 10
before substantial rod degeneration has taken place. Since mg of diaminobenzidine Sigma. After washing with PBS
in the central nervous system CNS, PSD-95 is known to to terminate the staining reactions, the sections were then
play an important role in synapse formation and or coverslipped with 50 glycerol in PBS.
maintenance, our results suggest that early loss of PSD-95 For fluorescence microscopy, the retinal sections were
from the rod terminals may be causally related to the incubated with mixed primary antibodies rabbit polyclonal
absence of rod–rod bipolar cell synapses in the rhodopsin anti-cone transducin antibody 1:1000 and mouse mono-
P347L transgenic retina. clonal anti-PSD-95 antibody 1:100 overnight at 48C. After
two washes in PBS for 15 min, sections were incubated with mixed secondary antibodies rhodamine-conjugated
2. Materials and methods goat anti-mouse immunoglobulin secondary antibody 1:50
