"Moonlighting" GAPDH Protein Localizes with AMPA Receptor GluA2 and L1 Axonal Cell Adhesion Molecule at Fiber Cell Borders in the Lens.

PURPOSE: The canonical role of glyceraldehyde phosphate dehydrogenase (GAPDH) is as an enzyme in glycolysis. GAPDH is also a principal "moonlighting" protein with additional roles at diverse sites in a variety of cells. Surface GAPDH on mammalian, yeast, and bacterial cells acts as a receptor and also mediates cell contacts. In neurons, extracellular GAPDH localizes at synapses. Two GAPDH binding partners at synapses are α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPA) GluA2 subunit at dendritic spines and L1 cell adhesion molecule at pre-synaptic membranes, and both proteins are also expressed in lenses. Fiber cell membrane protrusions and dendritic spines have similar size, shape, and spacing, contain F-actin, and express clathrin/AP-2 Adaptor at their surfaces linked with Tyr-phosphatase STEP-regulated endocytosis of AMPA/GluA2 receptors. AMPA receptors work with NMDA (N-methyl-d-aspartate) and GABA (γ-aminobutyric acid) receptors, calcium calmodulin kinase II (CaMKIIα), channel proteins, STEP, and ephrin receptors, which are also expressed in lenses. In neurons, coordinate AMPA/GluA2 receptor endocytosis with GAPDH is linked with disease. GAPDH was previously characterized as a fiber cell membrane protein and shown to decrease substantially in interior fiber cells in human age-related cataract. Here, we examined GAPDH spatial expression in healthy lenses in two vertebrate species. METHODS: In situ methods were used to examine GAPDH expression in lenses of healthy young adult rabbits and chickens. Immunoblots were used to detect L1 in lenses. RESULTS: The present study demonstrated that GAPDH is present at fiber cell borders in adult rabbit and chicken lenses with evidence of focal concentrations along the fiber cell perimeter, and overlapped with detection of p-Tyr-GluA2, L1, STEP, actin and clathrin. We observed that L1-140 kDa was the prominent form in lens. CONCLUSIONS: Our findings indicate investigations into GAPDH "moonlighting" activities similar to its role in cell-cell interactions at neuron surfaces are warranted in the lens.

Fragile X Syndrome FMRP Co-localizes with Regulatory Targets PSD-95, GABA Receptors, CaMKIIα, and mGluR5 at Fiber Cell Membranes in the Eye Lens.

Fmr1 and FMRP underlie Fragile X Syndrome (FXS) and are linked with related autism spectrum disorders (ASD). Fmr1 also has an essential role in eye and lens development. Lenses express FMRP along with γ-aminobutyric acid (GABA) receptors (GABARs), post-synaptic density protein 95 (PSD-95), Tyr-phosphatase STEP, CaMKIIα and Alzheimer's disease Aß precursor protein, which are verified targets of FMRP regulation in neurons and outline major topics in FXS/ASD research. PSD-95 as well as CaMKIIα transcripts undergo polypryimidine tract binding protein dependent alternative splicing in lens, consistent with PSD-95 translation in lens. At least 13 GABAR subunits and GAD25/65/67 GABA metabolism enzymes are expressed in lenses beginning in embryonic development, matching neural development. Interestingly, GABAergic drugs (e.g. baclofen) studied as FXS/ASD therapeutics are shown to resolve developmental vision defects in experimental myopia. Here, we demonstrated that FMRP co-localizes at fiber cell membranes with PSD-95, GABAAδ, GABAAß3, GABBR1, STEP, CaMKIIα, and mGluR5 in young adult lenses. GAD65 and GABA detection was greatest at the peri-nuclear lens region where fiber cell terminal differentiation occurs. These findings add to an extensive list of detailed parallels between fiber cell and neuron morphology and their lateral membrane spine/protrusions, also reflected in the shared expression of genes involved in the morphogenesis and function of these membrane structures, and shared use of associated regulatory mechanisms first described as distinguishing the neuronal phenotype. Future studies can determine if GABA levels currently studied as a FXS/ASD biomarker in the brain, and generated by GAD25/65/67 in a comparable cell environment in the lens, may be similarly responsive to Fmr1 mutation in lens. The present demonstration of FMRP and key regulatory targets in the lens identifies a potential for the lens to provide a new research venue, in the same individual, to inform about Fmr1/FMRP pathobiology in brain as well as lens.

PTBP-dependent PSD-95 and CamKIIα alternative splicing in the lens.

PURPOSE: Parallels described between neurons and lens fiber cells include detailed similarities in sub-cellular structures that increasingly show shared expression of genes involved in the construction and function of these structures in neurons. Intriguingly, associated modes of molecular regulation of these genes that had been thought to distinguish neurons have been identified in the lens as well. Both elongated cell types form membrane protrusions with similar size, shape, and spacing that exclude microtubules, contain F-actin, and are coated with the clathrin/AP-2 adaptor. Lenses express glutamate and gamma-aminobutyric acid (GABA) receptors with signaling and channel proteins shown to act together at neuronal membranes. Postsynaptic density protein 95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα) expression and functions illustrate the integration of aspects of neuronal molecular and cell biology and were investigated here in the lens. METHODS: Immunofluorescence, immunoblot, and RT-PCR methods were used to assess protein expression and alternative transcript splicing. RESULTS: We showed the essential dendritic spine scaffold protein PSD-95 is expressed in lenses and demonstrated lens PSD-95 transcripts undergo polypyrimidine tract binding protein (PTBP)-dependent alternative splicing of its pivotal exon 18 required to avoid nonsense-mediated decay, and showed PTBP-dependent alternative splicing of CaMKIIα transcripts in the lens. The PSD-95 protein was observed at fiber cell membranes overlapping with N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate and GABA receptor proteins, tyrosine phosphatase STEP, CaMKIIα, the Ca(V)1.3 calcium channel, and clathrin, which were previously identified at lens fiber cell membranes. During neurogenesis, miR-124 is expressed that suppresses PTBP1 and promotes these splicing events. miR-124 is also expressed in mammalian lenses and upregulated during lens regeneration in amphibians, consistent with previous demonstrations of PTBP1,2 and PTBP-dependent PTBP2 exon 10 splicing in rodent lenses. CONCLUSIONS: Findings of this dendritic spine scaffold protein and conservation of its key mode of molecular regulation in the lens provides further evidence that key aspects of the neuron morphogenetic program are shared with the lens.

NMDA glutamate receptor NR1, NR2A and NR2B expression and NR2B Tyr-1472 phosphorylation in the lens.

Detailed parallels described between lens fiber cell and neuron morphology, sub-cellular structure, and molecular biology include striking similarities in the ultrastructure of their vesicle transport machinery and the membrane protrusions that occur along the lateral surfaces of both cell types. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate receptor (NMDA) glutamate receptors (AMPARs/NMDARs) are the predominant receptors in neurons. These receptors have fundamental roles in neuron morphogenesis as well as neuron physiology and dynamic cell signaling, and specifically at dendritic spines. As a result, AMPAR and NMDAR dysregulation underlies several primary neural disorders that have also shown epidemiological associations with cataract. Previously, we demonstrated AMPAR GluA1 and REST (RE-1 silencing transcription factor)-regulated GluA2 subunits are expressed in the lens, and showed C-terminal phospho-tyrosine-GluA2, and striatal-enriched tyrosine phosphatase (STEP), as well as GluA2 Q/R RNA editing in lenses similar to neurons. Here, we demonstrated that REST-regulated NMDAR NR1, NR2A, and NR2B are also expressed in lenses and localize predominantly in fiber cell membranes, consistent with REST transcription factors, as well as miR-124 and other REST gene targets identified in the lens. We also showed NR2B Tyr-1472 phosphorylation occurs in lens. These p-Tyr-GluA2 and p-Tyr-NR2B phosphorylation events are linked with membrane insertion regulated by STEP. We next determined that NR1 transcripts that include exon 5 are produced in lens consistent with Fox-1 RNA binding protein isoforms linked with this alternative splicing event, and shown to be expressed in lens as well as brain. These findings provide further evidence that fundamental neuronal morphogenetic programs, and hallmark neuronal gene expression and modes of regulation, are shared with elongated fiber cells of the lens.