Elucidation of the interaction proteome of mitochondrial chaperone Hsp78 highlights its role in protein aggregation during heat stress.

Chaperones of the Hsp100/Clp family represent major components of protein homeostasis, conferring maintenance of protein activity under stress. The ClpB-type members of the family, present in bacteria, fungi, and plants, are able to resolubilize aggregated proteins. The mitochondrial member of the ClpB family in Saccharomyces cerevisiae is Hsp78. Although Hsp78 has been shown to contribute to proteostasis in elevated temperatures, the biochemical mechanisms underlying this mitochondria-specific thermotolerance are still largely unclear. To identify endogenous chaperone substrate proteins, here, we generated an Hsp78-ATPase mutant with stabilized substrate-binding behavior. We used two stable isotope labeling-based quantitative mass spectrometry approaches to analyze the role of Hsp78 during heat stress-induced mitochondrial protein aggregation and disaggregation on a proteomic level. We first identified the endogenous substrate spectrum of the Hsp78 chaperone, comprising a wide variety of proteins related to metabolic functions including energy production and protein synthesis, as well as other chaperones, indicating its crucial functions in mitochondrial stress resistance. We then compared these interaction data with aggregation and disaggregation processes in mitochondria under heat stress, which revealed specific aggregation-prone protein populations and demonstrated the direct quantitative impact of Hsp78 on stress-dependent protein solubility under different conditions. We conclude that Hsp78, together with its cofactors, represents a recovery system that protects major mitochondrial metabolic functions during heat stress as well as restores protein biogenesis capacity after the return to normal conditions.

Dissecting Alzheimer's disease pathogenesis in human 2D and 3D models.

The incidence of Alzheimer's disease is increasing with the aging population, and it has become one of the main health concerns of modern society. The dissection of the underlying pathogenic mechanisms and the development of effective therapies remain extremely challenging, also because available animal and cell culture models do not fully recapitulate the whole spectrum of pathological changes. The advent of human pluripotent stem cells and cell reprogramming has provided new prospects for tackling these challenges in a human and even patient-specific setting. Yet, experimental modeling of non-cell autonomous and extracellular disease-related alterations has remained largely inaccessible. These limitations are about to be overcome by advances in the development of 3D cell culture systems including organoids, neurospheroids and matrix-embedded 3D cultures, which have been shown to recapitulate extracellular pathologies such as plaque formation in vitro. Recent xenograft studies have even taken human stem cell-based disease modeling to an in vivo scenario where grafted neurons are probed in a disease background in the context of a rodent brain. Here, we review the latest developments in this emerging field along with their advantages, challenges, and future prospects.

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53.

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

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 Alzheimer's 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 Aß 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.

Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome.

Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40 years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n=29 control and n=41 DS). By ELISA, we quantified soluble and insoluble Aß40 and Aß42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Aß) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Aß40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.

Biliverdin reductase--a protein levels and activity in the brains of subjects with Alzheimer disease and mild cognitive impairment.

Biliverdin reductase-A is a pleiotropic enzyme involved not only in the reduction of biliverdin-IX-alpha into bilirubin-IX-alpha, but also in the regulation of glucose metabolism and cell growth secondary to its serine/threonine/tyrosine kinase activity. Together with heme oxygenase, whose metabolic role is to degrade heme into biliverdin-IX-alpha, it forms a powerful system involved in the cell stress response during neurodegenerative disorders. In this paper, an up-regulation of the biliverdin reductase-A protein levels was found in the hippocampus of the subjects with Alzheimer disease and arguably its earliest form, mild cognitive impairment. Moreover a significant reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A was found, and this was paralleled by a marked reduction in its reductase activity. Interestingly, the levels of both total and phosphorylated biliverdin reductase-A were unchanged as well as its enzymatic activity in the cerebella. These results demonstrated a dichotomy between biliverdin reductase-A protein levels and activity in the hippocampus of subjects affected by Alzheimer disease and mild cognitive impairment, and this effect likely is attributable to a reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A. Consequently, not just the increased levels of biliverdin reductase-A, but also its changed activity and phosphorylation state, should be taken into account when considering potential biomarkers for Alzheimer disease and mild cognitive impairment.

Atorvastatin treatment in a dog preclinical model of Alzheimer's disease leads to up-regulation of haem oxygenase-1 and is associated with reduced oxidative stress in brain.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Only acetylcholinesterase inhibitors and the NMDA antagonist memantine are approved for AD treatment. Recent preclinical and epidemiological studies proposed statins as novel therapeutics for AD, but the mechanisms of action are still unknown. Here, we demonstrate that atorvastatin (80 mg/d for 14.5 months) treatment resulted in an up-regulation of the inducible isoform of haem oxygenase (HO-1), an enzyme with significant neuroprotective activity. Atorvastatin selectively increased HO-1 in the parietal cortex but not cerebellum. In contrast, HO-2 was increased in cerebellum but not parietal cortex. No changes were observed in HO-1 or HO-2 in the liver. Significant negative correlations between HO-1 and oxidative stress indices and positive correlations with glutathione levels in parietal cortex were found. HO-1 up-regulation significantly correlated with lower discrimination learning error scores in aged beagles. Reference to therapeutic applications of atorvastatin in AD is discussed.

Long-term high-dose atorvastatin decreases brain oxidative and nitrosative stress in a preclinical model of Alzheimer disease: a novel mechanism of action.

Alzheimer disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss, inability to perform the activities of daily living and personality changes. Unfortunately, drugs effective for this disease are limited to acetylcholinesterase inhibitors that do not impact disease pathogenesis. Statins, which belong to the class of cholesterol-reducing drugs, were proposed as novel agents useful in AD therapy, but the mechanism underlying their neuroprotective effect is still unknown. In this study, we show that atorvastatin may have antioxidant effects, in aged beagles, that represent a natural higher mammalian model of AD. Atorvastatin (80 mg/day for 14.5 months) significantly reduced lipoperoxidation, protein oxidation and nitration, and increased GSH levels in parietal cortex of aged beagles. This effect was specific for brain because it was not paralleled by a concomitant reduction in all these parameters in serum. In addition, atorvastatin slightly reduced the formation of cholesterol oxidation products in cortex but increased the 7-ketocholesterol/total cholesterol ratio in serum. We also found that increased oxidative damage in the parietal cortex was associated with poorer learning (visual discrimination task). Thus, a novel pharmacological effect of atorvastatin mediated by reducing oxidative damage may be one mechanism underlying benefits of this drug in AD.

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways.

A neuropathological characteristic of Alzheimer's disease is the extracellular accumulation of amyloid beta peptide (Abeta) in neuritic plaques. Recent evidences suggested that soluble Abeta oligomers are the predominant neurotoxic species for neurons. Thus, considerable attention has been paid to discriminate the cytotoxic pathways of Abeta pre-fibrillar aggregates and mature fibrils. We showed that the mechanisms by which Abeta oligomers and fibrils generated reactive oxygen species differ in terms of site of production and kinetics, suggesting the involvement of different intra/extracellular pathways.

Wild type but not mutant APP is involved in protective adaptive responses against oxidants.

This study points out different behaviour between HEK cells overexpressing wild-type or mutant APP when exposed to oxidative insult. Although apparently both APPwt and APPmut overexpression conferred resistance to oxidative insult, some differences in terms of degree of protection was observed in the two clones. We found that the two clones differed, especially, in terms of redox profile. HEK-APPmut cells were characterized by higher levels of oxidative markers in comparison with HEK-APPwt. In addition, SOD activity appeared more efficient in HEK-APPwt than in HEK-APPmut, thus justifying the differences in terms of cell survival in the two clones. We suggest that, according to "hormesis theory", in HEK-APPwt cells low amount of oxidative stress can exert a beneficial effect that at a higher intensity results harmful. In contrast, HEK-APPmut cells lost this stress resistance probably because the degree of oxidative stress is too high and the antioxidant enzymes are themselves compromised.

Glutathionylation of the pro-apoptotic protein p53 in Alzheimer's disease brain: implications for AD pathogenesis.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. The exact mechanism for the AD pathogenesis is not clearly understood. However, a number of hypotheses have been proposed to explain the pathogenesis of AD. One the hypotheses is the oxidative stress hypothesis that is supported by a number of studies which reported an increase in the levels of reactive oxygen/reactive nitrogen species and their products with a concomitant decrease in the levels of antioxidant enzymes in AD brain. In the present study, we measured in AD brain the expression levels of different forms (monomer, dimer and tetramer) of the pro-apoptotic protein, p53, and observed greater levels of p53 monomer and dimer in AD brain compared to control. In addition, we also showed the selective glutathionylation of monomeric and dimeric form of p53 in AD brain. We propose that glutathionylation of p53 may prevent the formation of tetramer, an aggregate form required for effective action of p53, and may be involved in oxidative stress conditions and neurodegeneration observed in this dementing disorder.

Mitochondria as Potential Targets in Alzheimer Disease Therapy: An Update.

Alzheimer disease (AD) is a progressive and deleterious neurodegenerative disorder that affects mostly the elderly population. At the moment, no effective treatments are available in the market, making the whole situation a compelling challenge for societies worldwide. Recently, novel mechanisms have been proposed to explain the etiology of this disease leading to the new concept that AD is a multifactor pathology. Among others, the function of mitochondria has been considered as one of the intracellular processes severely compromised in AD since the early stages and likely represents a common feature of many neurodegenerative diseases. Many mitochondrial parameters decline already during the aging, reaching an extensive functional failure concomitant with the onset of neurodegenerative conditions, although the exact timeline of these events is still unclear. Thereby, it is not surprising that mitochondria have been already considered as therapeutic targets in neurodegenerative diseases including AD. Together with an overview of the role of mitochondrial dysfunction, this review examines the pros and cons of the tested therapeutic approaches targeting mitochondria in the context of AD. Since mitochondrial therapies in AD have shown different degrees of progress, it is imperative to perform a detailed analysis of the significance of mitochondrial deterioration in AD and of a pharmacological treatment at this level. This step would be very important for the field, as an effective drug treatment in AD is still missing and new therapeutic concepts are urgently needed.

Oxidative Stress in Neurodegenerative Diseases: From a Mitochondrial Point of View.

Age is the main risk factor for a number of human diseases, including neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, which increasing numbers of elderly individuals suffer. These pathological conditions are characterized by progressive loss of neuron cells, compromised motor or cognitive functions, and accumulation of abnormally aggregated proteins. Mitochondrial dysfunction is one of the main features of the aging process, particularly in organs requiring a high-energy source such as the heart, muscles, brain, or liver. Neurons rely almost exclusively on the mitochondria, which produce the energy required for most of the cellular processes, including synaptic plasticity and neurotransmitter synthesis. The brain is particularly vulnerable to oxidative stress and damage, because of its high oxygen consumption, low antioxidant defenses, and high content of polyunsaturated fats very prone to be oxidized. Thus, it is not surprising the importance of protecting systems, including antioxidant defenses, to maintain neuronal integrity and survival. Here, we review the role of mitochondrial oxidative stress in the aging process, with a specific focus on neurodegenerative diseases. Understanding the molecular mechanisms involving mitochondria and oxidative stress in the aging and neurodegeneration may help to identify new strategies for improving the health and extending lifespan.

γ-Oryzanol Improves Cognitive Function and Modulates Hippocampal Proteome in Mice.

Rice (Oryza sativa L.) is the richest source of γ-oryzanol, a compound endowed with antioxidant and anti-inflammatory properties. γ-Oryzanol has been demonstrated to cross the blood-brain barrier in intact form and exert beneficial effects on brain function. This study aimed to clarify the effects of γ-oryzanol in the hippocampus in terms of cognitive function and protein expression. Adult mice were administered with γ-oryzanol 100 mg/kg or vehicle (control) once a day for 21 consecutive days following which cognitive behavior and hippocampal proteome were investigated. Cognitive tests using novel object recognition and Y-maze showed that long-term consumption of γ-oryzanol improves cognitive function in mice. To investigate the hippocampal proteome modulated by γ-oryzanol, 2D-difference gel electrophoresis (2D-DIGE) was performed. Interestingly, we found that γ-oryzanol modulates quantitative changes of proteins involved in synaptic plasticity and neuronal trafficking, neuroprotection and antioxidant activity, and mitochondria and energy metabolism. These findings suggested γ-oryzanol as a natural compound able to maintain and reinforce brain function. Although more intensive studies are needed, we propose γ-oryzanol as a putative dietary phytochemical for preserving brain reserve, the ability to tolerate age-related changes, thereby preventing clinical symptoms or signs of neurodegenerative diseases.

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease.

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

Effects of oxidative and nitrosative stress in brain on p53 proapoptotic protein in amnestic mild cognitive impairment and Alzheimer disease.

Many studies reported that oxidative and nitrosative stress might be important for the pathogenesis of Alzheimer's disease (AD) beginning with arguably the earliest stage of AD, i.e., as mild cognitive impairment (MCI). p53 is a proapoptotic protein that plays an important role in neuronal death, a process involved in many neurodegenerative disorders. Moreover, p53 plays a key role in the oxidative stress-dependent apoptosis. We demonstrated previously that p53 levels in brain were significantly higher in MCI and AD IPL (inferior parietal lobule) compared to control brains. In addition, we showed that in AD IPL, but not in MCI, HNE, a lipid peroxidation product, was significantly bound to p53 protein. In this report, we studied by means of immunoprecipitation analysis, the levels of markers of protein oxidation, 3-nitrotyrosine (3-NT) and protein carbonyls, in p53 in a specific region of the cerebral cortex, namely the inferior parietal lobule, in MCI and AD compared to control brains. The focus of these studies was to measure the oxidation and nitration status of this important proapoptotic protein, consistent with the hypothesis that oxidative modification of p53 could be involved in the neuronal loss observed in neurodegenerative conditions.

IMiQ: a novel protein quality control compartment protecting mitochondrial functional integrity.

Aggregation processes can cause severe perturbations of cellular homeostasis and are frequently associated with diseases. We performed a comprehensive analysis of mitochondrial quality and function in the presence of aggregation-prone polypeptides. Despite a significant aggregate formation inside mitochondria, we observed only a minor impairment of mitochondrial function. Detoxification of aggregated reporter polypeptides as well as misfolded endogenous proteins inside mitochondria takes place via their sequestration into a specific organellar deposit site we termed intramitochondrial protein quality control compartment (IMiQ). Only minor amounts of endogenous proteins coaggregated with IMiQ deposits and neither resolubilization nor degradation by the mitochondrial protein quality control system were observed. The single IMiQ aggregate deposit was not transferred to daughter cells during cell division. Detoxification of aggregates via IMiQ formation was highly dependent on a functional mitochondrial fission machinery. We conclude that the formation of an aggregate deposit is an important mechanism to maintain full functionality of mitochondria under proteotoxic stress conditions.

Role of Mitochondrial Protein Quality Control in Oxidative Stress-induced Neurodegenerative Diseases.

Proteins are constantly exposed to environmental stressors such as free radicals and heat shock leading to their misfolding and later to aggregation. In particular mitochondrial proteins are challenged by reactive oxygen species (ROS) due to the oxidative metabolism of the organelle. Protein aggregation has been associated with a wide variety of pathological conditions called proteopathies. However, for the maintenance of protein and cellular homeostasis, mitochondria have developed an elaborate protein quality control system consisting of chaperones and ATP-dependent proteases, specifically employed to rescue this organelle from damage due to the accumulation of misfolded proteins and toxic aggregates. Aging is characterized by a general decline of mitochondrial functions, correlating with a decrease in mitochondrial protein quality control activity and an increase of free radical production. In particular in age-related diseases like neurodegeneration, a correlation between mitochondrial damage and disease onset has been established. In this review we summarize the current knowledge about mitochondrial protein quality control mechanisms in mammalian cells, with a special emphasis on the role in oxidative stress and in neurodegenerative diseases.

Amyloid ß-peptides interfere with mitochondrial preprotein import competence by a coaggregation process.

Aß peptides play a central role in the etiology of Alzheimer disease (AD) by exerting cellular toxicity correlated with aggregate formation. Experimental evidence has shown intraneuronal accumulation of Aß peptides and interference with mitochondrial functions. Nevertheless, the relevance of intracellular Aß peptides in the pathophysiology of AD is controversial. Here we found that the two major species of Aß peptides, in particular Aß42, exhibited a strong inhibitory effect on the preprotein import reactions essential for mitochondrial biogenesis. However, Aß peptides interacted only weakly with mitochondria and did not affect the inner membrane potential or the structure of the preprotein translocase complexes. Aß peptides significantly decreased the import competence of mitochondrial precursor proteins via an extramitochondrial coaggregation mechanism. Coaggregation and import inhibition were significantly stronger for the longer peptide Aß42, correlating with its importance in AD pathology. Our results demonstrate that direct interference of aggregation-prone Aß peptides with mitochondrial protein biogenesis represents a crucial aspect of the pathobiochemical mechanisms contributing to cellular damage in AD.