Chiara Cini

Elevated protein-bound levels of the lipid peroxidation product, 4-hydroxy-2-nonenal, in brain from persons with mild cognitive impairment

Oxidative damage is a feature of many age-related neurodegenerative diseases, including Alzheimers disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the free radical-mediated lipid peroxidation of unsaturated lipids, particularly arachidonic acid, in cellular membranes. In the present study we show for the first time in brain obtained at short postmortem intervals that the levels of HNE are elevated in mild cognitive impairment (MCI) hippocampus and inferior parietal lobules compared to those of control brain. Thus, increased levels of HNE in MCI brain implicate lipid peroxidation as an early event in AD pathophysiology and also suggest that the pharmacologic intervention to prevent lipid peroxidation at the MCI stage or earlier may be a promising therapeutic strategy to delay or prevent progression to AD.

Proteomic Analysis of Protein Expression and Oxidative Modification in R6/2 Transgenic Mice A Model of Huntington Disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are α-enolase, γ-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

CATECHOLAMINES OXIDATION BY XANTHINE OXIDASE

Dopamine and structurally related catecholamines in the presence of hydrogen peroxide are oxidized in vitro by xanthine oxidase producing the corresponding melanin pigments. The kinetic parameters of the reaction, measured as aminochrome formation, have been calculated. The rate of peroxidation depends on enzyme and hydrogen peroxide concentration. The optimum pH for the peroxidative activity of the enzyme is around 8.5. Activation of the peroxidative reaction is also elicited by catechol compounds through a redox cycle mechanism. Implications about the possible biochemical relevance of xanthine oxidase activity on catecholamines oxidation are discussed.

In vivo protective effects of ferulic acid ethyl ester against amyloid-beta peptide 1-42-induced oxidative stress.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid‐beta peptide (Aβ), a peptide that as both oligomers and fibrils is believed to play a central role in the development and progress of AD by inducing oxidative stress in brain. Therefore, treatment with antioxidants might, in principle, prevent propagation of tissue damage and neurological dysfunction. The aim of the present study was to investigate the in vivo protective effect of the antioxidant compound ferulic acid ethyl ester (FAEE) against Aβ‐induced oxidative damage on isolated synaptosomes. Gerbils were injected intraperitoneally (i.p.) with FAEE or with dimethylsulfoxide, and synaptosomes were isolated from the brain. Synaptosomes isolated from FAEE‐injected gerbils and then treated ex vivo with Aβ1–42 showed a significant decrease in oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3‐nitrotyrosine levels), and lipid peroxidation (4‐hydroxy‐2‐nonenal levels). Consistent with these results, both FAEE and Aβ1–42 increased levels of antioxidant defense systems, evidenced by increased levels of heme oxygenase 1 and heat shock protein 72. FAEE led to decreased levels of inducible nitric oxide synthase. These results are discussed with potential therapeutic implications of FAEE, a brain accessible, multifunctional antioxidant compound, for AD involving modulation of free radicals generated by Aβ.

Redox proteomic analysis of carbonylated brain proteins in mild cognitive impairment and early Alzheimer's disease.

Previous studies indicated increased levels of protein oxidation in brain from subjects with Alzheimers disease (AD), raising the question of whether oxidative damage is a late effect of neurodegeneration or precedes and contributes to the pathogenesis of AD. Hence, in the present study we used a parallel proteomic approach to identify oxidatively modified proteins in inferior parietal lobule (IPL) from subjects with mild cognitive impairment (MCI) and early stage-AD (EAD). By comparing to age-matched controls, we reasoned that such analysis could help in understanding potential mechanisms involved in upstream processes in AD pathogenesis. We have identified four proteins that showed elevated levels of protein carbonyls: carbonic anhydrase II (CA II), heat shock protein 70 (Hsp70), mitogen-activated protein kinase I (MAPKI), and syntaxin binding protein I (SBP1) in MCI IPL. In EAD IPL we identified three proteins: phosphoglycerate mutase 1 (PM1), glial fibrillary acidic protein, and fructose bisphospate aldolase C (FBA-C). Our results imply that some of the common targets of protein carbonylation correlated with AD neuropathology and suggest a possible involvement of protein modifications in the AD progression.

Redox proteomics in aging rat brain: Involvement of mitochondrial reduced glutathione status and mitochondrial protein oxidation in the aging process

Increasing evidence supports the notion that increased oxidative stress is a fundamental cause in the aging process and in neurodegenerative diseases. As a result, a decline in cognitive function is generally associated with brain aging. Reactive oxygen species (ROS) are highly reactive intermediates, which can modify proteins, nucleic acids, and polyunsaturated fatty acids, leading to neuronal damage. Because proteins are major components of biological systems and play key roles in a variety of cellular functions, oxidative damage to proteins represents a primary event observed in aging and age‐related neurodegenerative disorders. In the present study, with a redox proteomics approach, we identified mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain regions, such as cortex and hippocampus, that are commonly affected by the aging process. In all brain regions examined, many of the identified proteins were energy‐related, such as pyruvate kinase, ATP synthase, aldolase, creatine kinase, and α‐enolase. These alterations were associated with significant changes in both cytosolic and mitochondrial redox status in all brain regions analyzed. Our finding is in line with current literature postulating that free radical damage and decreased energy production are characteristic hallmarks of the aging process. In additon, our results further contribute to identifying common pathological pathways involved both in aging and in neurodegenerative disease development.

Proteomics-determined differences in the Concanavalin-A-fractionated proteome of hippocampus and inferior parietal lobule in subjects with Alzheimer’s disease and mild cognitive impairment: Implications for progression of AD

Alzheimers disease (AD) is the most common type of dementia, comprising 60-80% of all reported cases, and currently affects 5.2 million Americans. AD is characterized pathologically by the accumulation of senile plaques (SPs), neurofibrillary tangles (NFTs), and synapse loss. The early stages of memory loss associated with AD have been studied in a condition known as amnestic mild cognitive impairment (MCI), arguably the earliest form of AD. In spite of extensive research across a variety of disciplines, the cause of AD remains elusive. Proteomics techniques have helped to advance knowledge about AD by identifying irregularities in protein expression and post-translational modifications (PTMs) in AD brain. Glycosylation is a less studied PTM with regards to AD and MCI. This PTM is important to study because glycosylation is involved in proper protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles, and these processes are impaired in AD. Concanavalin-A (Con-A) binds to N-linked glycoproteins, but hydrophobic sites on nonglycoproteins are also known to bind Con-A. To our knowledge, the present study is the first to examine Con-A-associated brain proteins in MCI and AD with focus on the hippocampus and inferior parietal lobule (IPL) brain regions. Proteins found in AD hippocampus with altered levels are glutamate dehydrogenase (GDH), glial fibrillary acidic protein (GFAP), tropomyosin 3 (TPM3), Rab GDP-dissociation inhibitor XAP-4 (XAP4), and heat shock protein 90 (HSP90). Proteins found with altered levels in AD IPL are alpha-enolase, gamma-enolase, and XAP-4. MCI hippocampal proteins with altered levels are dihydropyrimidase-2 (DRP2), glucose-regulated protein 78 (GRP-78), protein phosphatase related protein Sds-22 (Sds22), and GFAP and the only protein found with altered levels in MCI IPL was beta-synuclein. These results are discussed with reference to biochemical and pathological alterations in and progression of AD.

Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimers disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.

Impairment of proteostasis network in Down syndrome prior to the development of Alzheimer's disease neuropathology: redox proteomics analysis of human brain.

DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimers disease neuropathology after the age of 40years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aβ 1-42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.

Neuropathological role of PI3K/Akt/mTOR axis in Down syndrome brain

Down syndrome (DS) is the most frequent genetic cause of intellectual disability characterized by the presence of three copies of chromosome 21 (Chr21). Individuals with DS have sufficient neuropathology for a diagnosis of Alzheimers disease (AD) after the age of 40years. The aim of our study is to gain new insights in the molecular mechanisms impaired in DS subjects that eventually lead to the development of dementia. We evaluate the PI3K/Akt/mTOR axis in the frontal cortex from DS cases (under the age of 40years) and DS with AD neuropathology compared with age-matched controls (Young and Old). The PI3K/Akt/mTOR axis may control several key pathways involved in AD that, if aberrantly regulated, affect amyloid beta (Aβ) deposition and tau phosphorylation. Our results show a hyperactivation of PI3K/Akt/mTOR axis in individuals with DS, with and without AD pathology, in comparison with respective controls. The PI3K/Akt/mTOR deregulation results in decreased autophagy, inhibition of IRS1 and GSK3β activity. Moreover, our data suggest that aberrant activation of the PI3K/Akt/mTOR axis acts in parallel to RCAN1 in phosphorylating tau, in DS and DS/AD. In conclusion, this study provides insights into the neuropathological mechanisms that may be engaged during the development of AD in DS. We suggest that deregulation of this signaling cascade is already evident in young DS cases and persist in the presence of AD pathology. The impairment of the PI3K/Akt/mTOR axis in DS population might represent a key-contributing factor to the neurodegenerative process that culminates in Alzheimer-like dementia.