Neurofilaments are the major neuronal target of hydroxynonenal-mediated protein cross-links.

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently binds amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated to be intimately associated with pathological lesions of Alzheimer's disease (AD), suggesting that oxidative stress is a major component in the disease. Here, we examined the HNE-cross-linking modifications by using an antibody specific for a lysine-lysine cross-link. Since in a prior study we noted no immunolabeling of neuritic plaques or neurofibrillary tangles but instead found strong labeling of axons, we focused this study on axons. Axonal labeling was examined in mouse sciatic nerve, and immunoblotting showed the cross-link was restricted to neurofilament heavy and medium subunits, which while altering migration, did not indicate larger NF aggregates, indicative of intermolecular cross-links. Examination of mice at various ages showed the extent of modification remaining relatively constant through the life span. These findings demonstrate lipid-cross-linking peroxidation primarily involves lysine-rich neurofilaments and is restricted to intramolecular cross-links.

A novel perspective on tau in Alzheimer's disease.

Mainstream thinking is dominated by the notion that the aggregation of specific proteins within neurons, and their subsequent formation into cytoplasmic and extracellular lesions, directly elicits neuronal dysfunction and death. Current dogma, for example, maintains that phosphorylated tau protein, the major component of neurofibrillary tangles, is a central mediator of disease pathogenesis. In this article, we challenge this classic notion by proposing that tau phosphorylation represents a compensatory response mounted by neurons against oxidative stress that serves a protective function. This concept provides a better understanding of the mechanisms underlying disease pathophysiology and also provides a window for therapeutic intervention.

Neuronal binucleation in Alzheimer disease hippocampus.

AIMS: The literature and teachings instruct that neurones in the adult brain are fully differentiated, quiescent cells that never divide. Somewhat surprisingly, and counter to such dogma, susceptible neurones in Alzheimer disease display an activated cell cycle phenotype. However, whether this leads to a coordinated procession through the cell cycle is unclear, particularly whether neurones enter anaphase and beyond. To begin to address this issue, in this study we sought to determine whether nuclear division occurs in these neurones. METHODS: We examined a series of 101 archived, routinely stained hippocampal sections collected at post mortem for neuropathological evaluation for evidence of neuronal binucleation. RESULTS: We report for the first time, binucleated neurones within the hippocampus in cases of Alzheimer disease but not in control cases (P < 0.05). CONCLUSIONS: While a relatively rare event, occurring once every 20,000 neurones, this morphological evidence that neuronal cells within the cortical regions of the adult human brain in Alzheimer disease contain two nuclei supports the hypothesis that neuronal cells can re-enter into a coordinated cell cycle that culminates in nuclear division.

Neuronal death and survival under oxidative stress in Alzheimer and Parkinson diseases.

Neuronal death is a common feature in neurodegenerative diseases including Alzheimer disease (AD) and Parkinson disease (PD). This occurs over years, not the minutes of classically defined apoptosis, and neurons show both responses of apoptosis and regeneration, evidenced by accumulated oxidative insult and attempts at cell cycle re-entry. There is recent evidence suggesting that several known gene mutations in causing familial AD (amyloid beta protein precursor, presenilin-1, or presenilin-2 gene) and familial PD (Parkin, PINK-1, or DJ-1 gene) are associated with increased oxidative stress. Also, several known genetic (e.g. Apolipoprotein Eepsilon4 variant) and environmental (e.g. metals or pesticides exposure) risk factors of sporadic AD and/or PD are associated with increased oxidative stress. In concord, patients at the preclinical stages of AD and PD as well as cellular and animal models of the diseases provide consistent evidence that oxidative insult is a significant early event in the pathological cascade of AD and PD. In contrast to the general aspects of the pathological hallmarks, aggregation of the disease-specific proteins such as amyloid-beta, tau, and alpha-synuclein may act as a compensatory (survival) response against the oxidative insult via the mechanism that the disease-specific structures sequester redox-active metals. Expanding knowledge of the molecular mechanisms of organism longevity indicates that pro-longevity gene products such as forkhead transcription factors and sirtuins are involved in the insulin-like signaling pathway and oxidative stress resistance against aging. An enhancement of the pro-longevity signaling (e.g. caloric restriction) may be a promising approach as anti-oxidative strategy against age-associated neurodegenerative diseases.

Sensitivity of 14-3-3 protein test varies in subtypes of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: The increase of the 14-3-3 protein in CSF is used as a diagnostic test in Creutzfeldt-Jakob disease (CJD), but the sensitivity and specificity of the 14-3-3 test are disputed. One reason for the dispute may be the recently established heterogeneity of sporadic CJD. The relationship between CSF 14-3-3 protein and sporadic CJD subtypes, distinguished by electrophoretic mobility of proteinase K-resistant prion protein (PrP(Sc)) and genotype at codon 129 of the prion protein gene, has not been elucidated. METHODS: The authors examined the 14-3-3 protein test in 90 patients with sporadic CJD. PrP(Sc) type (type 1 or type 2) and the genotype at polymorphic codon 129 were determined in each patient. Mutations were excluded by prion gene sequencing. RESULTS: The authors' findings indicate that the sensitivity of the 14-3-3 test is higher in patients with molecular features of the classic sporadic CJD than in patients with the nonclassic CJD subtypes. The difference appears to be related to the PrP(Sc) type and not to the codon 129 genotype. Disease duration before 14-3-3 testing might also have an influence because it was shorter in classic sporadic CJD. CONCLUSION: The Creutzfeldt-Jakob disease clinical subtype should be considered when interpreting results of the 14-3-3 test.

Pathobiology of familial hypercholesterolemic atherosclerosis.

Many factors play a role in the development of atherosclerotic lesions. One of the leading risk factors for development of atherosclerosis is familial hypercholesterolemia (FH). FH is a genetic disease characterized by a deficiency, and/or mutation, of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells (EC), a high level of low density lipoprotein in the plasma, and early, spontaneous development of atherosclerosis and skin xanthoma. In this review we describe Watanabe heritable hyperlipidemic (WHHL) rabbits, which represent such an animal model for human FH. This strain of the rabbits is characterized by a genetic deficiency or mutation of functional LDL receptors and develops severe atherosclerosis, which is pathologically similar to familial homozygous hyperlipidemic patients. The most completely characterized animal model is the Watanabe rabbit, a model of homozygous and heterozygous type IIa hypercholesterolemia related to an LDL receptor deficiency. Additional manipulation such as aortic injury in this rabbit model induces the development of atherosclerotic lesions that are structurally similar to those found in humans. Thus, this model of hypercholesterolemia fulfils the above criteria set, i.e. it is able to provide new insights for a better understanding of the pathogenesis of atherosclerosis and for testing new treatment strategies.

Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the 'two hit' hypothesis.

There are multiple lines of evidence showing that oxidative stress and aberrant mitogenic signaling play an important role in the pathogenesis of Alzheimer disease. However, the chronological relationship between these and other events associated with disease pathogenesis is not known. Given the important role that mitogen-activated protein kinase (MAPK) pathways play in both mitogenic signaling (ERK) and cellular stress signaling (JNK/SAPK and p38), we investigated the chronological and spatial relationship between activated ERK, JNK/SAPK and p38 during disease progression. While all three kinases are activated in the same susceptible neurons in mild and severe cases (Braak stages III-VI), in non-demented cases with limited pathology (Braak stages I and II), both ERK and JNK/SAPK are activated but p38 is not. However, in non-demented cases lacking any sign of pathology (Braak stage 0), either ERK alone or JNK/SAPK alone can be activated. Taken together, these findings indicate that MAPK pathways are differentially activated during the course of Alzheimer disease and, by inference, suggest that both oxidative stress and abnormalities in mitotic signaling can independently serve to initiate, but both are necessary to propagate, disease pathogenesis. Therefore, we propose that both 'hits', oxidative stress and mitotic alterations, are necessary for the progression of Alzheimer disease.

Active glycation in neurofibrillary pathology of Alzheimer disease: N(epsilon)-(carboxymethyl) lysine and hexitol-lysine.

Advanced glycation end products are a diverse class of posttranslational modifications, stemming from reactive aldehyde reactions, that have been implicated in the pathogenesis of a number of degenerative diseases. Because advanced glycation end products are accelerated by, and result in formation of, oxygen-derived free radicals, they represent an important component of the oxidative stress hypothesis of Alzheimer disease (AD). In this study, we used in situ techniques to assess N(epsilon)-(Carboxymethyl)lysine (CML), the predominant advanced glycation end product that accumulates in vivo, along with its glycation-specific precursor hexitol-lysine, in patients with AD as well as in young and aged-matched control cases. Both CML and hexitol-lysine were increased in neurons, especially those containing intracellular neurofibrillary pathology in cases of AD. The increase in hexitol-lysine and CML in AD suggests that glycation is an early event in disease pathogenesis. In addition, because CML can result from either lipid peroxidation or advanced glycation, while hexitol-lysine is solely a product of glycation, this study, together with studies demonstrating the presence of 4-hydroxy-2-nonenal adducts and pentosidine, provides evidence of two distinct oxidative processes acting in concert in AD neuropathology. Our findings support the notion that aldehyde-mediated modifications, together with oxyradical-mediated modifications, are critical pathogenic factors in AD.

Hibernation, a model of neuroprotection.

Hibernation, a natural model of tolerance to cerebral ischemia, represents a state of pronounced fluctuation in cerebral blood flow where no brain damage occurs. Numerous neuroprotective aspects may contribute in concert to such tolerance. The purpose of this study was to determine whether hibernating brain tissue is tolerant to penetrating brain injury modeled by insertion of microdialysis probes. Guide cannulae were surgically implanted in striatum of Arctic ground squirrels before any of the animals began to hibernate. Microdialysis probes were then inserted in some animals after they entered hibernation and in others while they remained euthermic. The brain tissue from hibernating and euthermic animals was examined 3 days after implantation of microdialysis probes. Tissue response, indicated by examination of hematoxylin and eosin-stained tissue sections and immunocytochemical identification of activated microglia, astrocytes, and hemeoxygenase-1 immunoreactivity, was dramatically attenuated around probe tracks in hibernating animals compared to euthermic controls. No difference in tissue response around guide cannulae was observed between groups. Further study of the mechanisms underlying neuroprotective aspects of hibernation may lead to novel therapeutic strategies for stroke and traumatic brain injury.

Abortive apoptosis in Alzheimer's disease.

Multiple studies suggest that neuronal death in Alzheimer's disease (AD) is the result of an apoptotic mechanism. However, the stereotypical manifestations that define the terminal phases of apoptosis, such as chromatin condensation, apoptotic bodies, and blebbing, are not seen in AD. In this study, we show that the caspases, such as caspase 6, which cleave amyloid-beta protein precursor (A beta PP) and presenilins, are localized to the pathological lesions associated with AD. However, while upstream caspases such as 8 and 9 are clearly found in association with the intraneuronal pathology in AD, downstream caspases such as 3, 6 and 7 are present only at control levels. Given that execution of apoptosis requires amplification of the caspase-mediated apoptotic signal, our results indicate that in AD there is a lack of effective apoptotic signal propagation to downstream caspase effectors. Therefore, while the presence of caspases, especially caspase 6, in association with extracellular deposits of amyloid-beta, could obviously have important ramifications on the proteolytic processing of A beta PP and, thereby, on disease pathogenesis, it seems that AD represents the first in vivo situation reported in which the initiation of apoptosis does not proceed to caspase-dependent cell death. This novel phenomenon of apoptotic avoidance, which we term abortive apoptosis, or abortosis, may represent an exit from the caspase-induced apoptotic program that leads to neuronal survival in AD.

Sequestration of iron by Lewy bodies in Parkinson's disease.

Central to the oxidative stress hypothesis of Parkinson's disease (PD) pathogenesis is the ability of iron to generate hydroxyl radicals via the Fenton reaction, and the consistent demonstration of iron elevation in the pars compacta region of the substantia nigra. However, uncertainty exists as to whether the excess iron exists in a state suitable for redox chemistry. Here, using a method we developed that detects redox-active iron in situ, we were able to demonstrate strong labeling of Lewy bodies in substantia nigra pars compacta neurons in PD. In contrast, cortical Lewy bodies in cases of Lewy body variant of Alzheimer's disease were unstained. While the presence of elevated iron in PD substantiates the oxidative stress hypothesis, one must remember that these are viable neurons, indicating that Lewy bodies may act to sequester iron in PD brains in a protective, rather than degenerative, mechanism. The absence of redox-active iron in neocortical Lewy bodies highlights a fundamental difference between cortical and brain stem Lewy bodies.

Evidence for a novel heme-binding protein, HasAh, in Alzheimer disease.

Multiple lines of evidence indicate that oxidative stress is an integral component of the pathogenesis of Alzheimer disease (AD). The precipitating cause of such oxidative stress may be misregulated iron homeostasis because there are profound alterations in heme oxygenase-1 (HO-1), redox-active iron, and iron regulatory proteins. In this regard, HasA, a recently characterized bacterial protein involved in heme acquisition and iron metabolism, may also be important in the generation of reactive oxygen species (ROS) given its ability to bind heme and render iron available for free radical generation through the Fenton reaction. To study further the role of heme binding and iron metabolism in AD, we show an abnormal localization of anti-HasA to the neurofibrillary pathology of AD, but not in normal-appearing neurons in the brains of cases of AD or in age-matched controls. These results suggest the increased presence in AD of a HasA homologue or protein sharing a common epitope with HasA, which we term HasAh. We conclude that heme binding of HasAh is a potential source of free soluble iron and therefore toxic free radicals in AD and in aging. This furthers the evidence that redox-active iron and subsequent Fenton reaction generating reactive oxygen are critical factors in the pathogenesis of AD.

Alexander disease: Alzheimer disease of the developing brain?

Alexander disease is a leukodystrophy-like neurodegenerative disease that typically presents in infancy or childhood. The disease is essentially a sporadic condition, and there is no known genetic predisposition or metabolic abnormality. The hallmark of the disease is the diffuse accumulation of Rosenthal fibers (RF) throughout the central nervous system. Although an etiological relationship of the RF to disease pathogenesis has been suspected since the initial description of Alexander disease, such a relationship has not been confirmed. We previously identified a number of oxidative post-translational modifications, including advanced glycation end products and lipid peroxidation adducts, in intimate association with the RF of Alexander disease. Such oxidative protein damage provides a mechanism, through protein crosslinking, for insolubility and accumulation of RF. Notably, these findings show a striking parallel with the biochemical features of age-related neurodegenerative diseases such as Alzheimer disease. Therefore, Alexander disease and Alzheimer disease likely share a common pathogenesis, namely oxidative injury as a potential primary process in the etiology and pathogenesis.

Is increased redox-active iron in Alzheimer disease a failure of the copper-binding protein ceruloplasmin?

One of the most striking features of Alzheimer disease (AD) is an accumulation of iron in neurofibrillary tangles and senile plaques. Intriguingly, this iron is found as both iron (II) and iron (III) and is redox-active. To address the issue of whether such iron participates in redox cycling, it was essential to investigate how iron (II) accumulates, since oxidation of iron (II) can lead to the generation of reactive oxygen species. To begin to address this issue, here we investigated ceruloplasmin, a key protein involved in the regulation of the redox state of iron by converting iron (II) to iron (III). Cases of AD and age-matched controls, obtained at autopsy with similar postmortem intervals, display similar levels of ceruloplasmin immunoreactivity that is mainly confined to neurons. However, in marked contrast, cases of AD show a significant increase in ceruloplasmin within the neuropil determined by immunoblot analysis of tissue homogenates as well as a generalized increased neuropil staining. Together, these findings suggest that neuronal induction of ceruloplasmin is feeble in AD, even while there is an increase in tissue ceruloplasmin. Therefore, a failure of neuronal ceruloplasmin to respond to iron may be an important factor that then leads to an accumulation of redox-active iron in neurons in AD.

Molecular genetic alterations in radiation-induced astrocytomas.

Astrocytic tumors occasionally arise in the central nervous system following radiotherapy. It is not clear if these gliomas represent a unique molecular genetic subset. We identified nine cases in which an astrocytoma arose within ports of previous radiation therapy, with total doses ranging from 2400 to 5500 cGy. Irradiated primary lesions included craniopharyngioma, pituitary adenoma, Hodgkin's lymphoma, ependymoma, pineal neoplasm, rhabdomyosarcoma, and three cases of lymphoblastic malignancies. Patients ranged from 9 to 60 years of age and developed secondary tumors 5 to 23 years after radiotherapy. The 9 postradiation neoplasms presented as either anaplastic astrocytoma (3 cases) or glioblastoma multiforme (6 cases). Two of the latter contained malignant mesenchymal components. We performed DNA sequence analysis, differential polymerase chain reaction (PCR), and quantitative PCR on DNA from formalin-fixed, paraffin-embedded tumors to evaluate possible alterations of p53, PTEN, K-ras, EGFR, MTAP, and p16 (MTS1/CDKN2) genes. By quantitative PCR, we found EGFR gene amplification in 2 of 8 tumors. One of these demonstrated strong immunoreactivity for EGFR. Quantitative PCR showed chromosome 9p deletions including p16 tumor suppressor gene (2 of 7 tumors) and MTAP gene (3 of 7). Five of 9 tumors demonstrated diffuse nuclear immunoreactivity for p53 protein. Sequencing of the p53 gene in these 9 cases revealed a mutation in only one of these cases, a G-to-A substitution in codon 285 (exon 8). Somewhat unexpectedly, no mutations were identified in PTEN, a commonly altered tumor suppressor gene in de novo glioblastoma multiformes. Unlike some radiation-induced tumors, no activating point mutations of the K-ras proto-oncogene or base pair deletions of tumor suppressor genes were noted. These radiation-induced tumors are distinctive in their high histological grade at clinical presentation. The spectrum of molecular genetic alterations appears to be similar to that described in spontaneous high grade astrocytomas, especially those of the de novo type.

Dimethylargininase, a nitric oxide regulatory protein, in Alzheimer disease.

In this study, we show that dimethylargininase, a zinc protein involved in the regulation of nitric oxide synthase, is specifically elevated in neurons displaying cytoskeletal abnormalities and oxidative stress in Alzheimer disease (AD) while none of this enzyme was found in neurons in age-matched control cases. Seen in the context of earlier studies showing widespread nitric oxide related damage in AD and the role of dimethylargininase to activate nitric oxide synthetase, through catalytic removal of its endogenous inhibitors, these findings indicate major alterations in nitric oxide regulation in AD. Further, that low levels of zinc specifically inhibit dimethylargininase may provide a link between the numerous studies showing specific abnormalities in zinc and oxidative stress. Finally, our results provide additional evidence that oxidative stress- and nitric oxide-mediated events play important roles in the pathogenesis of AD.

Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the mechanism.

Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface. The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 33, 6301-6309]. Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations. The primary difference in the two groups is the loop conformation of residue Pro 402, which is serine in muscle PK. In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure. The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model. The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other. To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted. Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effectors, while the mutant Y443F was essentially equivalent to wild-type muscle PK protein except for a lower affinity to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.

Radiologic and pathologic findings of intracerebral schwannoma.

We report the radiologic and pathologic findings of an intracerebral schwannoma. MR imaging studies showed a superficially located cystic mass with an enhancing nodule and evidence of peritumoral edema or gliosis.