Glaucoma is characterized by retinal ganglion cell (RGC) pathology and a progressive loss of vision. Previous studies suggest RGC death is responsible for vision loss in glaucoma, yet evidence from other neurodegenerative diseases suggests axonal degeneration, in the absence of neuronal loss, can significantly affect neuronal function. To characterize RGC degeneration in the DBA/2 mouse model of glaucoma, we quantified RGCs in mice of various ages using neuronal-specific nuclear protein (NeuN) immunolabeling, retrograde labeling, and optic nerve axon counts. Surprisingly, the number of NeuN-labeled RGCs did not decline significantly until 18 months of age, at which time a significant decrease in RGC somal size was also observed. Axon dysfunction and degeneration occurred before loss of NeuN-positive RGCs, because significant declines in RGC number assayed by retrograde tracers and axon counts were observed at 13 months. To examine whether axonal dysfunction/degeneration affected gene expression in RGC axons or somas, NeuN and neurofilament-heavy (NF-H) immunolabeling was performed along with quantitative reverse transcription-PCR for RGC-specific genes in retinas of aged DBA/2 mice. Although these mice had similar numbers of NeuN-positive RGCs, the expression of neurofilament light, Brn-3b, and Sncg mRNA varied; this variation in RGC-specific gene expression was correlated with the appearance of NF-H immunoreactive RGC axons. Together, these data support a progression of RGC degeneration in this model of glaucoma, beginning with loss of retrograde label, where axon dysfunction and degeneration precede neuronal loss. This progression of degeneration suggests a need to examine the RGC axon as a locus of pathology in glaucoma.

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, ␤'-coatomer protein and G s ␤, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (K a approximately 7 pM) when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.Modulation of the levels of the second messenger cAMP in cells is important in the regulation of numerous physiological processes, including those in the immune/inflammatory systems, vascular smooth muscle, and the brain. Cyclic nucleotide phosphodiesterases (PDEs) 1 are a diverse family of enzymes that hydrolyze cAMP and cGMP and thus play an important role in modulating cAMP levels (1). The cAMP-specific phosphodiesterases (PDE4s) can be differentiated from other PDEs by sequence homology of the catalytic region of the enzymes (2) and by their ability to be specifically inhibited by the drug rolipram. Rolipram and other specific PDE4 inhibitors have been shown to have anti-depressant, anti-inflammatory, and smooth muscle relaxant activity in humans (2). The PDE4 enzymes are also characterized by the presence of unique regions of amino acid sequence outside the catalytic region of the proteins, which are called upstream conserved regions 1 and 2 (UCR1 and UCR2) and are located in the amino-terminal half of the proteins (3). The PDE4s are comprised of a large family of isoforms, encoded by four different genes (PDE4A, PDE4B, PDE4C, and PDE4D) in humans, with additional diversity being generated by alternative mRNA splicing (2). We and other groups (3-5) have characterized five different isoforms encoded by the human PDE4D gene, all of which appear to be conserved among mammals (6, 7). The five isoforms differ by the substitution of unique blocks of amino acids at the...

Little is known about molecular changes occurring within retinal ganglion cells (RGCs) before their death in glaucoma. Taking advantage of the fact that ␥-synuclein (Sncg) mRNA is expressed specifically and highly in adult mouse RGCs, we show in the DBA/2J mouse model of glaucoma that there is not only a loss of cells expressing this gene, but also a downregulation of gene expression of Sncg and many other genes within large numbers of RGCs. This downregulation of gene expression within RGCs occurs together with reductions in FluoroGold (FG) retrograde transport. Surprisingly, there are also large numbers of Sncg-expressing cells without any FG labeling, and among these many that have a marker previously associated with disconnected RGCs, accumulation of phosphorylated neurofilaments in their somas. These same diseased retinas also have large numbers of RGCs that maintain the intraocular portion while losing the optic nerve portion of their axons, and these disconnected axons terminate within the optic nerve head. Our data support the view that RGC degeneration in glaucoma has two separable stages: the first involves atrophy of RGCs, whereas the second involves an insult to axons, which causes the degeneration of axon portions distal to the optic nerve head but does not cause the immediate degeneration of intraretinal portions of axons or the immediate death of RGCs.

These observations suggest that in glaucoma, retina and optic nerve head microglia activation may be a factor in the early decline in function of the optic nerve and its subsequent degeneration.

Changes in microglial cell activation and distribution are associated with neuronal decline in the CNS, particularly under pathological conditions. Activated microglia converge on the initial site of axonal degeneration in human glaucoma, yet, their part in its pathophysiology remains unresolved. To begin with, it is unknown whether microglia activation precedes or is a late consequence of retinal ganglion cell (RGC) neurodegeneration. Here, we address this critical element in DBA/2J (D2) mice, an established model of chronic inherited glaucoma, using as a control the congenic substrain DBA/2J Gpnmb+/SjJ (D2G), which is not affected by glaucoma. We analyzed the spatial distribution and timecourse of microglial changes in the retina, as well as within the proximal optic nerve prior to and throughout ages when neurodegeneration has been reported. Exclusively in D2 mice, we detected early microglia clustering in the inner central retina and unmyelinated optic nerve regions, with microglia activation peaking by 3 months of age. Between 5 and 8 months of age, activated microglia persisted and concentrated in the optic disc, but also localized to the retinal periphery. Collectively, our findings suggest microglia activation is an early alteration in the retina and optic nerve in D2 glaucoma, potentially contributing to disease onset or progression. Ultimately, detection of microglial activation may have value in early disease diagnosis, while modulation of microglial responses may alter disease progression.