Bert C. Lynn

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

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.

Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinsons disease and Alzheimers 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 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.

Increased levels of 4-hydroxynonenal and acrolein, neurotoxic markers of lipid peroxidation, in the brain in Mild Cognitive Impairment and early Alzheimer's disease

Previous studies show increased levels of lipid peroxidation and neurotoxic by-products of lipid peroxidation including 4-hydroxynonenal (HNE) and acrolein in vulnerable regions of the Alzheimers disease (AD) brain. To determine if lipid peroxidation occurs early in progression of AD, we analyzed levels of HNE and acrolein in the hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyrus (SMTG) and cerebellum (CER) of 7 subjects with Mild Cognitive Impairment (MCI), six subjects with early AD (EAD) and sevem age-matched control subjects using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Our data show that there is a statistically significant (P<0.05) increase in HNE in HPG, SMTG and CER in MCI compared to age-matched control subjects. Specimens of SMTG also showed a significant increase in levels of acrolein in MCI. Comparison of EAD and control subjects showed a statistically significant increase in HNE in HPG and SMTG and a significant increase in acrolein in all three brain regions studied. We did not observe any statistically significant differences between MCI and EAD specimens. These results suggest that lipid peroxidation occurs early in the pathogenesis of AD.

Proteomic identification of proteins specifically oxidized by intracerebral injection of amyloid β-peptide (1–42) into rat brain: Implications for Alzheimer’s disease

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimers disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain

The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and alpha-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, alpha-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

Proteomic identification of proteins oxidized by Aβ(1–42) in synaptosomes: Implications for Alzheimer's disease

Protein oxidation has been implicated in Alzheimers disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, alpha-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1-42 (Abeta 1-42) leads to the formation of protein carbonyls. In order to test the involvement of Abeta(1-42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Abeta(1-42) treatment leads to oxidatively modified proteins, consistent with the notion that Abeta(1-42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified beta-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Abeta(1-42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, gamma-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD.

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders.

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimers disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins

Ubiquitin carboxyl‐terminal hydrolase L‐1 (UCH L‐1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L‐1 as one specific target of protein oxidation in Alzheimers disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L‐1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L‐1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor α/ATP synthase and neurofilament‐L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.

Identification of Nucleophosmin as an NF-κB Co-activator for the Induction of the Human SOD2 Gene *

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-κB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-κB affinity chromatography to isolate potential NF-κB interacting proteins. Proteins eluted from the NF-κB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-κB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-κB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines.

Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Aβ(1–42): Implications for Alzheimer's disease

Protein oxidation has been shown to lead to loss of protein function, increased protein aggregation, decreased protein turnover, decreased membrane fluidity, altered cellular redox poteintial, loss of Ca2+ homeostaisis, and cell death. There is increasing evidence that protein oxidation is involved in the pathogenesis of Alzheimers disease and amyloid beta-peptide (1-42) has been implicated as a mediator of oxidative stress in AD. However, the specific implications of the oxidation induced by Abeta(1-42) on the neurodegeneration evident in AD are unknown. In this study, we used proteomic techniques to identify specific targets of oxidation in transgenic Caenorhabditis elegans (C. elegans) expressing human Abeta(1-42). We identified 16 oxidized proteins involved in energy metabolism, proteasome function, and scavenging of oxidants that are more oxidized compared to control lines. These results are discussed with reference to Alzheimers disease.