The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O À:2 leads to formation of peroxynitrite ONOO -, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration. Keywords: Alzheimer's disease, neurodegeneration, 3-nitrotyrosine, proteomics, reactive nitrogen species. 2 leads to formation of peroxynitrate ONOO ) , which, following protonation, generates cytotoxic species that oxidize and nitrate proteins (Beckman 1996). (Formally, the NO 2 added to tyrosine is a nitrite, not nitrate, but the literature has consistently used the latter term, so we shall as well in this paper.)The more common amino acidic targets of oxidation are lysine, histidine, cysteine and methionine (Butterfield and Stadtman 1997), whereas tyrosine is the commonly nitrated amino acid (Souza et al. 2001). In particular, nitration of tyrosine residues is a formal oxidation (Butterfield and Stadtman 1997), a chemical modification that has been used to investigate the mechanism by which tyrosine residues Received November 11, 2002; revised manuscript received February 19, 2003; accepted February 20, 2003. Address correspondence and reprint requests to Professor D. Allan Butterfield, Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA. E-mail: dabcns@uky.eduAbbreviations used: AD, Alzheimer's disease; HCNP, hippocampal cholinergic neurostimulating peptide; IPL, inferior parietal lobule; 3NT, 3-nitrotyrosine; PEBP, phosphatidylethanolamine-binding protein; PBST, phosphate-buffered saline with 0.01% sodium azide and 0.2% Twe...

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Defects in assembly are suggested to signal the dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. Weak mutant alleles of SPP382 (also called NTR1) were found to suppress defects in two proteins required for spliceosome activation, Prp38p and Prp8p. Spp382p is shown necessary for cellular splicing, with premRNA and, for some alleles, excised intron, accumulating after inactivation. Like spp382-1, a mutant allele of AAR2 was identified in this suppressor screen. Like Spp382p, Aar2p has a reported role in spliceosome recycling and is found with Spp382p in a complex recovered with a mutant version of the spliceosomal core protein Prp8p. Possible insight into to the spp382 suppressor phenotype is provided by the observation that defective splicing complexes lacking the 5 exon cleavage intermediate are recovered by a tandem affinity purification-tagged Spp382 derivative. Stringent proteomic and two-hybrid analyses show that Spp382p also interacts with Cwc23p, a DNA J-like protein present in the spliceosome and copurified with the Prp43p DExD͞H-box ATPase. Spp382p binds Prp43p and Prp43p requires Spp382p for intron release from the spliceosome. Consistent with a related function in the removal of defective complexes, three prp43 mutants are also shown to suppress splicing defects, with efficiencies inversely proportionate to the measured ATPase activities. These and related genetic data support the existence of a Spp382p-dependent turnover pathway acting on defective spliceosomes.pre-mRNA ͉ RNA processing ͉ spp382 ͉ small nuclear ribonucleoprotein ͉ Prp43 S pliceosome assembly in Saccharomyces cerevisiae (yeast) extract progresses through the sequential addition of the U1, U2, and U4͞U6.U5 small nuclear ribonucleoprotein (snRNP) particles. The snRNP-complete complex then undergoes conformational changes to form a catalytically active enzyme (reviewed in refs. 1 and 2). In this hallmark activation step, the extensively paired U4͞U6 snRNAs unwind, and the U6 snRNA displaces U1 at the 5Ј splice site and associates with the branchpoint region through U2 snRNA contacts. The U1 and U4 snRNAs dissociate from (or more weakly bind) the mature spliceosome, and neither is required for catalysis. Although alternate models have been proposed e.g., ref. 3, the isolation of cellular U2, U5, and U6 snRNP complexes (4, 5) strongly supports an equivalent spliceosome maturation pathway in vivo. This view is reinforced by studies of cotranscriptional premRNA splicing that reveal striking similarities in the timing of splicing factor association in vivo and in vitro (6, 7).The specificity determinants for U4͞U6.U5 tri-snRNP recruitment to the prespliceosome are unclear, although U5 snRNP-exon interactions and transient U4 and U6 snRNA contacts with the premRNA 5Ј splice site region may contribute (1,8,9). Once bound, the U5 snRNP-associated Prp28p and Brr2p DExD͞H-box proteins are critical for U1 and U4 snRNP release from the spliceosome, respectively (10...

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