Rapid glutamate receptor 2 trafficking during retinal degeneration
© Lin et al; licensee BioMed Central Ltd. 2012
Received: 27 November 2011
Accepted: 10 February 2012
Published: 10 February 2012
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© Lin et al; licensee BioMed Central Ltd. 2012
Received: 27 November 2011
Accepted: 10 February 2012
Published: 10 February 2012
Retinal degenerations, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are characterized by photoreceptor loss and anomalous remodeling of the surviving retina that corrupts visual processing and poses a barrier to late-stage therapeutic interventions in particular. However, the molecular events associated with retinal remodeling remain largely unknown. Given our prior evidence of ionotropic glutamate receptor (iGluR) reprogramming in retinal degenerations, we hypothesized that the edited glutamate receptor 2 (GluR2) subunit and its trafficking may be modulated in retinal degenerations.
Adult albino Balb/C mice were exposed to intense light for 24 h to induce light-induced retinal degeneration (LIRD). We found that prior to the onset of photoreceptor loss, protein levels of GluR2 and related trafficking proteins, including glutamate receptor-interacting protein 1 (GRIP1) and postsynaptic density protein 95 (PSD-95), were rapidly increased. LIRD triggered neuritogenesis in photoreceptor survival regions, where GluR2 and its trafficking proteins were expressed in the anomalous dendrites. Immunoprecipitation analysis showed interaction between KIF3A and GRIP1 as well as PSD-95, suggesting that KIF3A may mediate transport of GluR2 and its trafficking proteins to the novel dendrites. However, in areas of photoreceptor loss, GluR2 along with its trafficking proteins nearly vanished in retracted retinal neurites.
All together, LIRD rapidly triggers GluR2 plasticity, which is a potential mechanism behind functionally phenotypic revisions of retinal neurons and neuritogenesis during retinal degenerations.
Retinal degenerations (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are progressive disorders initiated by photoreceptor stress and are accelerated by photoreceptor death, which effectively deafferents the inner retina and evolves into formal retinal remodeling [1–3]. Thus, retinal remodeling proceeds through three phases: 1, photoreceptor stress; 2, photoreceptor death and 3, complex neural remodeling . Two of the major hallmarks of retinal remodeling are growth of novel neurites and functional reprogramming of existing retinal neurons [1–8]. Pathogenic neuronal reprogramming and de novo neuritogenesis are not isolated to retinal tissues, as pathological revision also occurs in neurodegenerative diseases such as epilepsy  and Alzheimer's disease . Retinal remodeling limits the effectiveness of vision rescue strategies including photoreceptor- and retinal pigment epithelium (RPE)-directed therapies [4, 6, 7, 11, 12]. Better understanding of the mechanisms underlying retinal remodeling will improve the outcomes of genetic, molecular, cellular and bionic rescues.
Deafferentation of the neural retina eliminates the intrinsic glutamatergic drive by the sensory retina  and induces glutamate receptor reprogramming before gross topologic restructuring of the retina begins [4, 13]. In phase 2 degenerating retinas with extensive rod death, the downstream rod-specific signaling pathways persists [13, 14], and bipolar cells still respond to glutamate receptor agonists [4, 7, 15]. Among the glutamate receptors (GluRs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate fast synaptic transmission at excitatory synapses in CNS and are tetrameric assemblies of subunits GluR1-4 encoded by separate genes . Their involvement and modulation during neuronal development, synaptic plasticity and structural remodeling is fundamental to timing and coherence of developing neural networks . In brain, combined neuronal activity and pathologic insults trigger rapid changes in postsynaptic AMPA receptor attributes (e.g. subunit composition) and may control Ca2+ permeability . Ca2+ fluxes play critical roles in neural function, including the regulation of neurite outgrowth and synaptogenesis , synaptic transmission and plasticity , and cell survival . GluR2 in heteromeric AMPARs renders the channel low permeable to Ca2+ [22, 23], so that even a modest alteration in the level of GluR2 is expected to have profound implications for synaptic efficacy and neuronal survival .
Given prior evidence of iGluR reprogramming in human RP and animal models of RD [4, 8, 25], we hypothesized that retinal iGluRs, especially GluR2 subunits are modulated in retinal degenerative diseases. GluR2 subunit expression is associated with vertical channel retinal processing [26–28], and its expression limits AMPAR permeability to Ca2+ . In this sense it is thought to be neuroprotective [30, 31]. To study the kinetics of GluR2 expression and trafficking in retinal degenerative disease, we used the LIRD model, which contains the full spectrum of sequelae found in naturally occurring and engineered forms of retinal degeneration and remodeling, including early retinal stress, photoreceptor loss, Müller cell remodeling, neuritogenesis , and remodeling of all neural cell populations in the retina and formation of microneuromas [8, 12]. Our analysis of glutamate receptors and neuritogenesis in the light-damage model spans phases 1 and 2. This work demonstrated that in a LIRD model, GluR2 levels and trafficking rapidly increased in response to light-induced photoreceptor stress and death, providing a potential feedback mechanism for controlling Ca2+ permeability in retinal neurons. Most importantly, GluR2 upregulation may occur in ON bipolar cells, which are normally hyperpolarized by glutamate. Expression of AMPA receptors would change their polarity as predicted by Marc et al 2007  and Jones et al.  in mouse, rabbit and human retina. In addition, the motor protein KIF3A colocalized well with PSD-95 and GRIP1 at novel sprouting neurites, potentially indicating a chaperone role for KIF3A, guiding GluR2 and its trafficking proteins to newly forming dendritic processes.
We reveal a correlation of neurite formation and retraction with changes in expression of GluR2 and GluR2 trafficking machinery in the LIRD model. Alterations in GluR2 expression may provide a feedback mechanism for altering Ca2+ permeability in deafferented retinal neurons, therefore be responsible for de novo neuritogenesis, and ultimately contribute to functional reprogramming of neural circuitry.
Normally, increasing the ratio of GluR2 subunits in iGluRs plays a protective role by decreasing Ca2+ loads in neurons, which likely can not be blocked by AMPA receptors inhibitor NBQX . Additionally, GluR2-mediated decreases in Ca2+ permeability can prevent GluR1 phosphorylation-dependent increases in calcium conductance . In LIRD, GluR1 expression is not changed while GluR1 phosphorylation decreases and levels of calcium-buffering proteins calretinin and calbindin remain stable. Similarly, data from diabetic subjects showed significant retinal increases of GluR2 but not GluR1 immunoreactivity in the OPL and IPL [53–55]. GluR2 upregulation in various retinal disease models suggests that this may be a protective response to photoreceptor-mediated stress and deafferentation.
Nevertheless, GluR2 levels (mRNA) were found unchanged in rd mouse retina . Most inherited degenerations are slow compared to light damage and their time profiles are complex [1, 2]. Even the fast rd model requires 3 months for all photoreceptors to die and there is no known coherent stress period (at least we don't know if anyone has published such an analysis). Light damage has a very precise onset of a few hours. The 1995 Duvoison paper  does not specify the time point at which the 35S GluR ISH autoradiographic measurements were made. As shown by Jones et al. [13, 57], Marc et al. [1, 2, 4, 12], RD (including light damage) have complex time profiles and pass through three distinct phases: phase 1 (photoreceptor stress and early death), phase 2 (active photoreceptor death), phase 3 (remodeling after all photoreceptors are lost). Our examination of that paper suggests that the rd animals were adult, in which case they were in late phase 3 remodeling and the outer retina was completely decimated, lacking bipolar cell and horizontal cell dendrites. The purpose of our current paper was to profile the kinetics of glutamate receptors before such a pathologic state. Indeed our results show that the major changes in GluR expression occur acutely during phase 1 of light damage and quickly normalize, suggesting that GluR2 modulation is acute.
GluR2-mediated changes in Ca2+ permeability may be also responsible for the subsequent neurite outgrowth of retinal neurons. In embryonic chick retinal neurons, early activation of Ca2+-permeable AMPA receptors reduces neurite outgrowth , and influx of Ca2+ through these channels could be responsible for the reduction in neurite outgrowth of cultured hippocampal pyramidal cells . GluR2 co-localizing with its trafficking proteins PICK1, ABP, GRIP1 and stargazin at new neurites during LIRD suggests that this same mechanism may be represented in deafferented retina. The interaction of stargazin with AMPA receptors is essential for delivering functional receptors to the surface membrane, whereas binding with PSD-95, a GluR-clustering molecule, is required for targeting the AMPA receptors to synapses . During GluR2 trafficking, the PICK1-ABP/GRIP interaction targets PICK1-PKCα complexes to ABP/GRIP-AMPA receptor complexes. GluR2 binds to PICK1 after phosphorylation at serine 880 by PKCα, which induces dissociation of GluR2 from GRIP and the subsequent internalization of GluR2 by PICK1 for recycling or degradation . Therefore, rapid alterations in GluR2 and its trafficking during early LIRD may reveal mechanisms to limit Ca2+-mediated damage or simply reflect loss of dendritic iGluRs as dendrites are lost [4, 13]. Normalization of GluR2 expression by pLX30 may reveal new GluR2 expression in novel dendrites that are in the process of forming new synaptic complexes.
This study demonstrates that expression of GluR2 and its trafficking proteins in the new dendrites may be guided through KIF3A, a member of the heteromeric family of kinesins which constitute a large family of microtubule motor proteins . In neurons, kinesin motors conduct transport to axons and neurotransmitter receptors to dendrites and GRIP1 directly interacts with kinesin enabling transport of dendritic proteins such as GluR2 . Our work reveals the expression of KIF3A in the OPL, where is dominated by ribbon synapses between presynaptic photoreceptors and postsynaptic horizontal cells and bipolar cells, both of which are involved in early reactive neuritogenesis in the degenerative retina. Interaction between KIF3A and GRIP1 and PSD-95 indicates that KIF3A has the potential to steer GRIP1 and PSD-95 to the new dendritic formation. Because GluR2 and its trafficking proteins are anchored with PSD-95, it is possible that GluR2 and its trafficking proteins are guided indirectly by PSD-95 and GRIP1 to the new processes.
In normal tissues, differential expression of GluRs by bipolar cells creates parallel ON and OFF channels in vision. ON bipolar cells express mGluR6 receptors while OFF bipolar cells express either AMPA or KA receptors. In retinal degenerative diseases, the observation of GluR2 in pathological newly-formed bipolar cell dendrites is a new finding and likely contributes to the functional reprogramming observed in bipolar cells of degenerative retina [4, 13]. Altered differential expression of GluRs by bipolar cells explains the phenotypic revision and reprogramming of bipolar cells observed in RD [7, 12, 13]. Previously, we demonstrated phenotypic plasticity in human RP and in animal models of cone-sparing RP where loss of rods, but not cones, leads to phenotype revision, inducing a switch from ON to OFF phenotypes in rod bipolar cells . Nevertheless, primate ON bipolar cells also express a cohort of AMPA receptor genes in normal retina  and GluR2 subunits have long been identified in rod bipolar cells [21, 26, 28, 55, 63]. The fact that normal rod bipolar cells express iGluR subunits argues that such switching may be within the normal capacity of bipolar cells .
In summary, this work is the first to study GluR2 and its trafficking proteins during RD. Our findings suggest that rapid alteration of GluR2 trafficking/regulation is involved in pathological neuritogenesis and is a potential mechanism behind phenotypic reprogramming of retinal neurons. Modulation of GluR2 expression and its trafficking may slow down primary neurite remodeling or limit neuritogenesis. However, further research is needed to separate the GluR2-mediated pathways that contribute to neuroprotection and pathological neuritogenesis.
Age-matched female Balb/C albino mice (The Jackson Laboratory, Bar Harbor, ME) were maintained in dim light (20-40 lux) on a 12-12 cycle in normal phase (lights on 7:00-19:00) with ad libitum access to food and water. All experimental procedures were designed to minimize animal number and suffering, and were conducted with approval of the Institutional Animal Care Committees at the University of Utah in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
LIRD and subretinal injections were performed as described previously . In brief, mice were placed into the light-damage chamber, where the visible light intensity ranged from 2,500 to 3,000 lux, for 24 h by excluding one normal night cycle. Following post-light exposure (pLX), all animals were returned to the dim cyclic light environment and maintained for 0, 1, 7, and 30 days (pLX0, pLX1, pLX7, and pLX30). Under ketamine-xylazine anesthesia, 5 μM NBQX (Sigma, St. Louis, MO, USA) and 50 μM D-AP5 (Sigma) were injected into the sub-retinal space (central region) of albino mice in 0.5 μl volumes using a 33-gauge micro syringe (Hamilton) 30 min before light exposure. A successful sub-retinal injection caused swelling of the retina. Control retinas were injected with vehicle (DMSO) (Sigma).
At the end of the experiment, eyes were rapidly harvested following decapitation under isoflurane anesthesia. For Western blotting analysis, retinas were dissected in Hank's balanced salt solution (HBSS, GIBCO, Carlsbad, CA, USA), then placed on ice in 1.5 ml tubes with RIPA buffer (RIPA lysis buffer kit, Santa Cruz, CA, USA) and homogenized using a sonicator (Fisher Scientific, Pittsburgh, PA, USA) three times for 12 sec each with 12 sec breaks between cycles. The samples were put on ice for 10 min, then centrifuged at 14,000 rpm at 4°C for 10 min. Supernatants were transferred to new tubes. The concentrations of protein in the samples were measured using the BCA assay (Pierce, Rockford, IL USA). For immunohistochemical analysis, whole eyes were removed and rinsed in HBSS, fixed in 4% paraformaldehyde (PFA) (Sigma) for 2 h at 4°C, and then washed with PBS (in g/L: NaCl 8, KCl 0.2, Na2HPO4 1.44, KH2PO4 0.24; pH 7.4) twice for 10 min each. Eyes were incubated in PBS with 20% sucrose for 4 h at 4°C. Serial 12 μm coronal sections were made with a cryostat microtome (Leica CM3050 S, Wetzlar, Germany) and collected on Superfrost/plus microscope slides (Fisher Scientific).
Retinas were lysed in RIPA buffer (RIPA lysis buffer kit, Santa Cruz) for 1 h at 4°C with gentle agitation. Lysates were immunoprecipitated for 1 h at 4°C using KIF3A antibody (1:1000) and then protein G agarose beads (Roche, Indianapolis, IN, USA) . Samples were analyzed by Western blotting.
Samples of entire group were pooled. Protein samples (~20 μg protein) were combined with NuPAGE LDS sample buffer (4×) (Invitrogen, Carlsbad, CA, USA) and NuPAGE sample reducing agent (10×) (Invitrogen), then boiled for 10 min at 95°C. Western blotting analysis was carried out using NuPAGE 4-12% Bis-Tris gels (Invitrogen) at 200 V for 40 min. Gels were electro-blotted onto PVDF membrane (Millipore, Bedford, MA, USA) for 1 h at 25 V using a wet electro-blotting system (XCell SureLock Mini-Cell, Invitrogen). Blots were blocked for 1 h in PBS with 0.1% Triton-x100, pH 7.4 (PBST) containing 5% non-fat dry milk (NFDM). Blots then were incubated overnight at 4°C in primary antibodies diluted in 5% NFDM-PBST solution (GluR2, 1:1000, Chemicon; pGluR2, 1:1000, Millipore; protein kinase C α (PKCα), 1:5000, Sigma; GluR1, 1:2000, Upstate; pGluR1, 1:1000, Millipore; PSD-95, 1:500, Chemicon; protein interacting with C kinase 1 (PICK1), 1:400, Santa Cruz; GRIP1, 1:1000, Millipore; stargazin, 1:500, Chemicon; AMPA receptor binding protein (ABP), 1:400, Santa Cruz; KIF3A, 1:1000, Covance; calretinin, 1:2000, Abcam; calbindin, 1:2000, Abcam; αCaMKII 1:1000, Upstate; βCaMKII: 1:1000, Invitrogen). Blots were washed three times for 10 min in PBST, incubated for 2 h in secondary antibodies (IgG-HRP, Santa Cruz; 1:5000 in 5% NFDM-PBST) followed by three more washes of 10 min in PBST. Immunostaining was revealed by the SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific, Waltham, MA, USA), and scanned using the Quantity One imaging system (Bio-Rad). Densitometry for each band was measured using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). β-actin (1:5000, Sigma) was used as a loading control.
Cryosections were washed 2 × 10 min in PBS, then blocked with blocking buffer for 30 min. Cryosections were incubated overnight at 4°C in primary antibodies diluted in blocking buffer (GluR2, 1:500, Chemicon or 1:200, Santa Cruz; PKCα, 1:2000; PSD-95, 1:500; PICK1, 1:200; GRIP1, 1:500; stargazin, 1:500; ABP, 1:100; KIF3A, 1:500; calbindin, 1:1000). These primary antibodies were from different species in every case and they did not cross-react on Western Blots. After washing 3 × 10 min in PBS, sections were incubated for 45 min at room temperature in secondary antibodies (cy3-, 488- or 647-conjugated IgG, Invitrogen) diluted 1:1000 in blocking buffer. For double or triple staining, sections were sequentially incubated with primary antibodies and secondary antibodies as above. After incubation with antibodies, sections were washed 3 × 10 min in PBS, then treated with 10 μM DAPI (Invitrogen) for 5 min at room temperature. Sections from all groups were processed simultaneously to reduce staining artifacts or intensity differences. Negative controls were performed by omission of the primary antibodies.
Fluorescent images were acquired with an Olympus FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). Settings were chosen so that pixel intensities for the brightest samples were just below saturation, except when the processes of retinal neurons had to be clearly determined, in which case signals from certain areas (soma of the retinal neurons) were saturated in order to obtain a clear perimeter of the neurites. Optical slice units were 0.5 μm. Neuritogenesis was analyzed as described . In brief, neurite length was measured from the point of emergence at the cell body to the tip of each segment. One section was selected from each animal, and 20 longest dendrites from 20 rod bipolar cells in a specific region (ventral mid-peripheral region, 120 μm in length) were measured and calculated as the mean. Quantification of the morphological parameters was carried out using ImageJ by investigators blinded to experimental conditions.
Data of neuritogenesis were expressed as mean ± SD and analyzed with SPSS 12.0 (SPSS Inc.). Statistical comparisons were made using Bonferroni tests and analysis of variance (ANOVA), P < 0.05 was defined as the level of significance. Protein levels represented pooled data of entire groups and were expressed as means only. To study the rationality of pooling samples in our experiment, we actually set up 3 batches of animals. There were 3 mice per group in each batch (i.e. 9 mice in total for each group). We pooled the 3 mice's retinas from each group in each batch and ran the western blotting for protein analysis. It was found that the biological variations for the 9 mice were low and GluR2 increase, for example, was significant during early LIRD (Additional file 1 Figure S1).
light-induced retinal degeneration
age-related macular degeneration
glutamate receptor 2
phosphorylation of glutamate receptor 2
Ca2+/calmodulin-dependent protein kinase II
protein kinase C α
glutamate receptor-interacting protein 1
postsynaptic density protein 95
kinesin family member 3A
AMPA receptor binding protein
protein interacting with C kinase 1
retinal pigment epithelium
outer plexiform layer
outer nuclear layer
inner nuclear layer
This project was supported by NIH EY002576 (REM), EY015128 (REM), EY014800 Vision Core (REM), Research to Prevent Blindness (REM), Signature Immunologics (REM), Edward N. and Della L. Thome Memorial Foundation grant for Age-Related Macular Degeneration Research (BWJ), a Research to Prevent Blindness Career Development Award (BWJ), Moran Eye Center Tiger Team Translational Medicine Award (BWJ), Fight For Sight (YHL, FRVC and WDF), Knights Templar Eye Foundation (YHL and FRVC), 5T32 HD07491 (FRVC), International Retinal Research Foundation (YHL, FRVC), and an unrestricted grant from Research to Prevent Blindness to the Moran Eye Center. We thank Kevin Rapp, Marguerite V. Shaw, Jia-Hui Yang, and Carl B. Watt for assistance on tissue handling and immunohistochemistry. We thank Dr. Changjiang Zou for assistance on immunoprecipitation.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.