Fragile X Mental Retardation Protein expression in the retina is regulated by light.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick.

Selective activation of mGluR8 receptors modulates retinal ganglion cell light responses.

Extracellular and whole-cell light-evoked responses of mouse retinal ganglion cells were recorded in the presence of the mGluR8 selective agonist, (S)-3,4-dicarboxy-phenylglycine (DCPG). Off-light responses were reversibly reduced in the presence of DCPG in wild-type but not in mGluR8-deficient retinas. On-responses were only marginally modulated by DCPG. During Off-responses, DCPG suppressed both excitatory and inhibitory synaptic conductances suggesting that mGluR8 receptor activity reduces glutamate release from bipolar cell terminals and possibly also the release of an inhibitory neurotransmitter from amacrine cell processes.

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors.

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.

Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model.

Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.

Inhibition of 15-lipoxygenase leads to delayed organelle degradation in the reticulocyte.

Mammalian cells are characterized by an endomembrane system. Nevertheless, some cells lose these membranes during their terminal differentiation, e.g. red blood cells and lens fiber cells of the eye. 15-Lipoxygenase is believed to be critical for this membrane degradation. Here we use cultivated rabbit reticulocytes in the presence or absence of a lipoxygenase inhibitor to provide further evidence for the importance of 15-lipoxygenase for the in vivo degradation of mitochondria. We find that inhibitor treatment retarded mitochondrial degradation, as shown by persistence of marker proteins and by direct visualization of mitochondria by electron microscopy.

The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia.

Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.

Opposing effects of protein kinase C and protein kinase A on metabotropic glutamate receptor signaling: selective desensitization of the inositol trisphosphate/Ca2+ pathway by phosphorylation of the receptor-G protein-coupling domain.

Signaling by the metabotropic glutamate receptor 1alpha (mGluR1alpha) can lead to the accumulation of inositol 1,4, 5-trisphosphate (InsP(3)) and cAMP and to the modulation of K(+) and Ca(2+) channel opening. At present, very little is known about how these different actions are integrated and eventually turned off. Unraveling the molecular mechanisms underlying these functions is crucial for understanding mGluR-mediated regulation of synaptic transmission. It has been shown that receptor-induced activation of the InsP(3) pathway is subject to feedback inhibition mediated by protein kinase C (PKC). In this study, we provide evidence for a differential regulation by PKC and protein kinase A of two distinct mGluR1alpha-dependent signaling pathways. PKC activation selectively inhibits agonist-dependent stimulation of the InsP(3) pathway but does not affect receptor signaling via cAMP. In contrast, protein kinase A potentiates agonist-independent signaling of the receptor via InsP(3). Furthermore, we demonstrate that the selectivity of PKC action on receptor signaling rests on phosphorylation of a threonine residue located in the G protein-interacting domain of the receptor. Modification at Thr(695) selectively disrupts mGluR1alpha-G(q/11) interaction without affecting signaling through G(s). Together, these data provide insight on the mechanisms by which selective down-regulation of a specific receptor-dependent signaling pathway can be achieved and on how cross-talk between different second messenger cascades may contribute to fine-tune short- and long-term receptor activity.

Cloning and immunocytochemical localization of a cyclic nucleotide-gated channel alpha-subunit to all cone photoreceptors in the mouse retina.

Cyclic nucleotide-gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3;,5;-cyclic adenosine monophosphate and 3;,5;-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ON-bipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide-gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC alpha-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide-gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision.

Retinal lesions induce differential changes in the expression of flip and flop isoforms of the glutamate receptor subunit GluR1 in the chick optic tectum.

A sensitive RNase protection assay was employed to determine the levels of mRNA encoding the GluR1 subunit flip and flop isoforms in the chick optic tectum and forebrain. We found that the flip GluR1 mRNA predominates in the forebrain, whereas the flop variant is more strongly expressed in the optic tectum. A temporal analysis of GluR1 variants in the embryonic and adult chick brain revealed that the flip isoform is more highly expressed at E12 than at P15-21, whereas mRNA levels of the flop isoform are higher at P15-21 than at E12. To study the effect of deafferentation on GluR1 expression, unilateral retinal lesions were performed. Two days later the mRNA levels of GluR1 flip and flop variants were decreased in the deafferented tectum, especially for the flop isoform. However, 7 days after the lesion, the mRNA levels of both GluR1 isoforms were increased, especially for the flip isoform. These results reveal an important control of the retinal input upon the expression of the different GluR1 isoforms. Furthermore, they indicate a differential spatial and temporal regulation of the flip and flop splice variants, suggesting the existence of a mechanism regulating differential splicing or possibly differential RNA stability.

Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth.

Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from post-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.

Differential localization of GABA(B) receptors in the mouse retina.

The mRNA distribution of the two cloned GABA(B) receptor variants, GABA(B)R1a and -R1b, was analysed in the retina by non-radioactive in situ hybridization. GABA(B)R1a transcripts were found in the inner nuclear and ganglion cell layers, probably in horizontal, amacrine and ganglion cells, whereas GABA(B)R1b transcripts were detected in the ganglion cell layer only. Together with a recent immunohistochemical localization of GABA(B)R1 in the retina, this indicates a differential targeting of the receptor variants to pre- and postsynaptic sites with GABA(B)R1a and -R1b localized to axonal and dendritic compartments, respectively. In this way, inhibition of neurotransmitter release and slow postsynaptic inhibition could be provided by receptor variants derived from the same gene.

A function for lipoxygenase in programmed organelle degradation.

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells of the eye lens, and keratinocytes. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.

Absence of antibodies to non-NMDA glutamate-receptor subunits in paraneoplastic cerebellar degeneration.

OBJECTIVE: To determine whether patients with antineuronal antibody-associated paraneoplastic cerebellar degeneration (PCD) harbor antibodies to non-N-methyl-D-aspartate glutamate receptors (GluRs) in their serum. BACKGROUND: A recent study identified antibodies to GluRs in the serum of patients with PCD. METHODS: Sera of 35 patients with PCD (20 anti-Yo, 5 anti-Ri, 5 anti-Tr, and 5 anti-Hu) were examined by immunohistochemistry and immunoblot techniques. The expression of GluRs was obtained after transfection of 293T cells with cDNA plasmids corresponding to GluR1, GluR2, GluR3, GluR4, GluR6, and GluR7. The tumors of four patients with anti-Yo-associated PCD and nine ovarian carcinomas from patients without PCD were examined for the expression of GluRs. RESULTS: The expression of each GluR subunit was confirmed using affinity-purified antibodies against the corresponding GluRs and with whole sera from two rabbits immunized with GluR3. Thirty-two sera from patients with PCD were negative and 3 showed equivocal reactivity with 293T cells expressing different GluRs. None of the 35 sera had a pattern of reactivity characteristic of any GluR antibody on immunohistochemistry of sections of rat brain. Eleven of 14 tumors did not express GluRs; only some cells of one paraneoplastic tumor expressed GluRs. CONCLUSIONS: That patients with antibody-associated PCD (anti-Yo, -Ri, -Tr, and -Hu) harbor GluR antibodies in their sera is unlikely. GluR antibodies cannot be used as markers of paraneoplastic neurologic disorders.

Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation.

On the basis of sequence homology and structural similarities, metabotropic glutamate receptors (mGluRs), extracellular Ca2+-sensing receptor, gamma-aminobutyric acid type B receptor, and pheromone receptors are enlisted in a distinct family within the larger G protein-coupled receptor superfamily. When expressed in heterologous systems, group I mGluRs can activate dual signal transduction pathways, phosphoinositides turnover and cAMP production. To investigate the structural basis of these coupling properties, we introduced single amino acid substitutions within the second and third intracellular loops (i2 and i3) of mGluR1alpha. Wild-type and mutant receptors were expressed in human embryonic kidney 293 cells and analyzed for their capacity to stimulate both signaling cascades. Each domain appeared to be critical for the coupling to phospholipase C and adenylyl cyclase. Within i2, Thr695, Lys697, and Ser702 were found to be selectively involved in the interaction with Gq class alpha subunit(s), whereas mutation of Pro698 and the deletion Cys694-Thr695 affected only Gs coupling. Furthermore, the mutation K690A profoundly altered mGluR1alpha signaling properties and imparted to the receptor the ability to couple to the inhibitory cAMP pathway. Within i3, we uncovered two residues, Arg775 and Phe781, that are crucial for coupling to both pathways, since their substitution leads to receptor inactivation.

The human metabotropic glutamate receptor 8 (GRM8) gene: a disproportionately large gene located at 7q31.3-q32.1.

Metabotropic glutamate receptors (GRMs), which constitute a family of genes, are neurotransmitter receptors that respond to glutamate stimulations by activating GTP-binding proteins and modulating second-messenger cascades. Pharmacological and expression studies of the rodent Grm8 gene suggest it could be a presynaptic receptor modulating glutamate release at the axon terminals. To study human GRM8, we have determined its nucleotide sequence and genomic organization. While the coding region of the gene spans only 2.3 kb, the gene encompasses approximately 1000 kb of DNA at the boundary of the q31.3-q32.1 bands of chromosome 7. This observation is relevant to the study of Smith-Lemli-Opitz syndrome and an autosomal dominant form of retinitis pigmentosa (RP10), since they map to the same region.

Expression of glutamate receptor subunit genes during development of the mouse retina.

The distribution of expression during retinal differentiation of nine AMPA and kainate glutamate receptor subunit genes was studied by in situ hybridization. Transcripts encoding all subunits were detected in the inner nuclear and ganglion cell layers of the developing mouse retina, but their time courses of expression differed. The earliest expression was detected at embryonic day 14 (E14) for GluR1, GluR4 and KA-2; all the other subunits were first detected at E16. Most subunits were expressed at higher levels during development than in the adult. This suggests they play a role in the processes of neuronal differentiation and synaptogenesis that occur during the early postnatal days in the rodent retina.

Localization of two metabotropic glutamate receptor genes, GRM3 and GRM8, to human chromosome 7q.

Metabotropic glutamate receptors (GRMs) are neurotransmitter receptors that respond to glutamate stimulations by activating GTP-binding proteins and modulating second-messenger cascades. Eight related GRMs have been identified to date. In this study, we have mapped GRM3 and GRM8 to human chromosome 7q21.1-q21.2 and 7q31.3-q32.1, respectively, using somatic cell hybrid and fluorescence in situ hybridization analysis. A yeast artificial chromosome contig was constructed surrounding the genes, allowing their location to be integrated into the genetic and physical map of chromosome 7.

A novel metabotropic glutamate receptor expressed in the retina and olfactory bulb.

A novel metabotropic glutamate receptor, mGluR8, was identified by screening a mouse retina cDNA library. This receptor is most related to mGluR4, mGluR7, and mGluR6 (74%, 74%, and 70% identical amino acid residues, respectively). Similar to these receptors, stimulation by L-glutamate or L-2-amino-4-phosphonobutyrate (L-APB) of Chinese hamster ovary (CHO) cells stably transfected with mGluR8 result in the inhibition of forskolin-stimulated adenylyl cyclase. In situ hybridization studies revealed a strong expression of the mGluR8 gene in the olfactory bulb, accessory olfactory bulb, and mammillary body. A weaker expression was found in the retina, and in scattered cells in the cortex and hindbrain. During development, the distribution of mGluR8 expression was more widespread. These results extend the diversity of metabotropic glutamate receptors in the CNS. Because at least two APB receptors are expressed in the retina, the use of this drug to block selectively the ON pathway needs to be reconsidered. The pharmacology and expression of mGluR8 in mitral/tufted cells suggest it could be a presynaptic receptor modulating glutamate release by these cells at their axon terminals in the entorhinal cortex.

Changes in expression of glutamate receptor subunits following photoreceptor degeneration in the rd mouse retina.

The effects of photoreceptor cell degeneration on the expression of nine alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/kainate glutamate receptor subunit genes were investigated in the rd mouse retina by in situ hybridization. All known AMPA/kainate receptor subunits were found to be expressed in normal mouse retina. Following retinal degeneration, the expression of KA-1 was reduced, that of the GluR7 subunit was greatly increased, and that of the other seven subunits were not significantly affected.