Proteomic analysis of brain proteins in APP/PS-1 human double mutant knock-in mice with increasing amyloid ß-peptide deposition: insights into the effects of in vivo treatment with N-acetylcysteine as a potential therapeutic intervention in mild cognitive impairment and Alzheimer's disease.

Proteomics analyses were performed on the brains of wild-type (WT) controls and an Alzheimer's disease (AD) mouse model, APP/PS-1 human double mutant knock-in mice. Mice were given either drinking water or water supplemented with N-acetylcysteine (NAC) (2 mg/kg body weight) for a period of five months. The time periods of treatment correspond to ages prior to Aß deposition (i.e. 4-9 months), resembling human mild cognitive impairment (MCI), and after Aß deposition (i.e. 7-12 months), more closely resembling advancing stages of AD. Substantial differences exist between the proteomes of WT and APP/PS-1 mice at 9 or 12 months, indicating that Aß deposition and oxidative stress lead to downstream changes in protein expression. Altered proteins are involved in energy-related pathways, excitotoxicity, cell cycle signaling, synaptic abnormalities, and cellular defense and structure. Overall, the proteomic results support the notion that NAC may be beneficial for increasing cellular stress responses in WT mice and for influencing the levels of energy- and mitochondria-related proteins in APP/PS-1 mice.

Potential in vivo amelioration by N-acetyl-L-cysteine of oxidative stress in brain in human double mutant APP/PS-1 knock-in mice: toward therapeutic modulation of mild cognitive impairment.

Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly. Although the underlying cause has yet to be established, numerous data have shown that oxidative stress is implicated in AD as well as in preclinical stages of AD, such as mild cognitive impairment (MCI). The oxidative stress observed in brains of subjects with AD and MCI may be due, either fully or in part, to increased free radicals mediated by amyloid-beta peptide (Abeta). By using double human mutant APP/PS-1 knock-in mice as the AD model, the present work demonstrates that the APP/PS-1 double mutation results in elevated protein oxidation (as indexed by protein carbonyls), protein nitration (as indexed by 3-nitrotyrosine), as well as lipid peroxidation (as indexed by protein-bound 4-hydroxy-2-nonenal) in brains of mice aged 9 months and 12 months. APP/PS-1 mice also exhibited lower levels of brain glutathione peroxidase (GPx) in both age groups studied, whereas glutathione reductase (GR) levels in brain were unaffected by the mutation. The activities of both of these antioxidant enzymes were significantly decreased in APP/PS-1 mouse brains, whereas the activity of glucose-6-phosphate dehydrogenase (G6PDH) was increased relative to controls in both age groups. Levels of peptidyl prolyl isomerase 1 (Pin1) were significantly decreased in APP/PS-1 mouse brain aged 9 and 12 months. Administration of N-acetyl-L-cysteine (NAC), a glutathione precursor, to APP/PS-1 mice via drinking water suppressed increased protein oxidation and nitration and also significantly augmented levels and activity of GPx in brain from both age groups. Oral administration of NAC also increased the diminished activity of GR and protected against lipid peroxidation in brains of 9-month-old APP/PS-1 mice only. Pin1 levels, GR levels, and G6PDH activity in brain were unaffected by oral administration of NAC in both age groups. These results are discussed with reference to the therapeutic potential of this brain-accessible glutathione precursor in the treatment of MCI and AD.

Association between male gender and cortical Lewy body pathology in large autopsy series.

Sex-linked factors may alter risk for neurodegenerative diseases. Definitive diagnoses are not established until autopsy, so neuropathological studies are critical. There have not been reported gender-related differences in neocortical Lewy bodies (LBs) using large multi-center autopsy series. We evaluated the associations between gender and pathologically characterized neurodegenerative diseases. Cases with Alzheimer's disease (AD), neocortical LBs, AD + neocortical LBs, or neither pathology were evaluated as separate groups. Results were corrected for possible confounders including age at death, smoking history, and education. The settings were the University of Kentucky Alzheimer's Disease Center and the National Alzheimer's Coordinating Center (NACC) Registry autopsy series; 3,830 subjects met inclusion criteria. Patients with neocortical ("diffuse") or intermediate ("limbic") LB pathologies tended to be male (male:female odds ratios ~2.9 with 95% CI 2.02-4.18). The preponderance of males dying with neocortical LB pathology was seen consistently across age groups and was not due to the potential confounders evaluated. By contrast, individuals dying with AD pathology were more likely to be female if dying over 80 (male:female odds ratio 0.66, 95% CI 0.50-0.88), but that tendency was not seen in individuals dying with AD pathology prior to age 80. Increased understanding of the male predominance in neocortical LB pathology may help guide clinicians, because males are more likely to be "undercalled" for neocortical LBs clinically, and females are more likely to be "overcalled" (P < 0.05 for both). Males are far more likely than females to die with neocortical LB pathology. This phenomenon may help guide medical practice including clinical trial study design.

Increased protein hydrophobicity in response to aging and Alzheimer disease.

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.

NOX activity is increased in mild cognitive impairment.

This study was undertaken to investigate the profile of NADPH oxidase (NOX) in the clinical progression of Alzheimer's disease (AD). Specifically, NOX activity and expression of the regulatory subunit p47phox and the catalytic subunit gp91phox was evaluated in affected (superior and middle temporal gyri) and unaffected (cerebellum) brain regions from a longitudinally followed group of patients. This group included both control and late-stage AD subjects, and also subjects with preclinical AD and with amnestic mild cognitive impairment (MCI) to evaluate the profile of NOX in the earliest stages of dementia. Data show significant elevations in NOX activity and expression in the temporal gyri of MCI patients as compared with controls, but not in preclinical or late-stage AD samples, and not in the cerebellum. Immunohistochemical evaluations of NOX expression indicate that whereas microglia express high levels of gp91phox, moderate levels of gp91phox also are expressed in neurons. Finally, in vitro experiments showed that NOX inhibition blunted the ability of oligomeric amyloid beta peptides to injure cultured neurons. Collectively, these data show that NOX expression and activity are upregulated specifically in a vulnerable brain region of MCI patients, and suggest that increases in NOX-associated redox pathways in neurons might participate in the early pathogenesis of AD.

Alterations of zinc transporter proteins ZnT-1, ZnT-4 and ZnT-6 in preclinical Alzheimer's disease brain.

Our previous studies demonstrate alterations of zinc (Zn) transporter proteins ZnT-1, ZnT-4 and ZnT-6 in vulnerable brain regions of subjects with mild cognitive impairment (MCI), and early and late stage Alzheimer's disease (AD), suggesting disruptions of Zn homeostasis may play a role in the pathogenesis of AD. A preclinical stage of AD (PCAD) has been described in which subjects show no overt clinical manifestations of AD, but demonstrate significant AD pathology at autopsy. To determine if alterations of ZnT proteins occur in PCAD, we measured ZnT-1, ZnT-4 and ZnT-6 in the hippocampus/parahippocampal gyrus (HPG) and cerebellum (CER) of seven PCAD subjects and seven age-matched normal control (NC) subjects using Western blot analysis and immunohistochemistry. Our results show a significant decrease (P < 0.05) of ZnT-1 in HPG of PCAD subjects, along with an increase of ZnT-4 in PCAD CER and ZnT-6 in PCAD HPG, but a significant decrease in PCAD CER compared to NC subjects. Confocal microscopy of representative sections of HPG shows altered ZnTs are associated with neurons immunopositive for MC-1, a monoclonal antibody that identifies neurons early in formation of neurofibrillary tangles. Overall, our results suggest that alterations in Zn transport proteins may contribute to the pathology observed in PCAD subjects before onset of clinical symptoms.

Evidence that gamma-secretase mediates oxidative stress-induced beta-secretase expression in Alzheimer's disease.

Beta-secretase (BACE1), an enzyme responsible for the production of amyloid beta-peptide (Abeta), is increased by oxidative stress and is elevated in the brains of patients with sporadic Alzheimer's disease (AD). Here, we show that oxidative stress fails to induce BACE1 expression in presenilin-1 (gamma-secretase)-deficient cells and in normal cells treated with gamma-secretase inhibitors. Oxidative stress-induced beta-secretase activity and sAPPbeta levels were suppressed by gamma-secretase inhibitors. Levels of gamma- and beta-secretase activities were greater in brain tissue samples from AD patients compared to non-demented control subjects, and the elevated BACE1 level in the brains of 3xTgAD mice was reduced by treatment with a gamma-secretase inhibitor. Our findings suggest that gamma-secretase mediates oxidative stress-induced expression of BACE1 resulting in excessive Abeta production in AD.

Redox proteomic identification of 4-hydroxy-2-nonenal-modified brain proteins in amnestic mild cognitive impairment: insight into the role of lipid peroxidation in the progression and pathogenesis of Alzheimer's disease.

Numerous investigations point to the importance of oxidative imbalance in mediating AD pathogenesis. Accumulated evidence indicates that lipid peroxidation is an early event during the evolution of the disease and occurs in patients with mild cognitive impairment (MCI). Because MCI represents a condition of increased risk for Alzheimer's disease (AD), early detection of disease markers is under investigation. Previously we showed that HNE-modified proteins, markers of lipid peroxidation, are elevated in MCI hippocampus and inferior parietal lobule compared to controls. Using a redox proteomic approach, we now report the identity of 11 HNE-modified proteins that had significantly elevated HNE levels in MCI patients compared with controls that span both brain regions: Neuropolypeptide h3, carbonyl reductase (NADPH), alpha-enolase, lactate dehydrogenase B, phosphoglycerate kinase, heat shock protein 70, ATP synthase alpha chain, pyruvate kinase, actin, elongation factor Tu, and translation initiation factor alpha. The enzyme activities of lactate dehydrogenase, ATP synthase, and pyruvate kinase were decreased in MCI subjects compared with controls, suggesting a direct correlation between oxidative damage and impaired enzyme activity. We suggest that impairment of target proteins through the production of HNE adducts leads to protein dysfunction and eventually neuronal death, thus contributing to the biological events that may lead MCI patients to progress to AD.

Oxidatively modified RNA in mild cognitive impairment.

Studies show increased oxidative damage in the brains of subjects with Alzheimer's disease (AD) and mild cognitive impairment (MCI). To determine if RNA oxidation occurs in MCI, sections of hippocampus/parahippocampal gyrus (HPG) from 5 MCI, 5 late stage AD (LAD) and 5 age-matched normal control (NC) subjects were subjected to immunohistochemistry using antibodies against 8-hydroxyguanine (8-OHG) and 1-N2-propanodeoxyguanosine (NPrG). Confocal microscopy showed 8-OHG and NPrG immunostaining was significantly (p<0.05) elevated in MCI and LAD HPG compared with NC subjects and was predominately associated with neurons identified using the MC-1 antibody that recognizes conformational alterations of tau, which are associated with early neurofibrillary tangle formation. Pretreating sections with RNase or DNase-I showed immunostaining for both adducts was primarily associated with RNA. In addition, levels of both adducts in MCI were comparable to those measured in LAD, suggesting RNA oxidation may be an early event in the pathogenesis of neuron degeneration in AD.

TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations.

Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal-dominant disorder caused by mutations in the gene valosin-containing protein (VCP). The CNS pathology is characterized by a novel pattern of ubiquitin pathology distinct from sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) without VCP mutations. TAR DNA binding protein 43 (TDP-43) was recently identified as a major disease protein in the ubiquitin-positive inclusions of sporadic and familial FTLD-U. To determine whether the ubiquitin pathology associated with mutations in VCP is characterized by the accumulation of TDP-43, we analyzed TDP-43 in the CNS pathology of five patients with VCP gene mutations. Accumulations of TDP-43 colocalized with ubiquitin pathology in inclusion body myopathy and Paget disease of bone, including both intranuclear inclusions and dystrophic neurites. Similar to FTLD-U, phosphorylated TDP-43 was detected only in insoluble brain extracts from affected brain regions. Identification of TDP-43, but not VCP, within ubiquitin-positive inclusions supports the hypothesis that VCP gene mutations lead to a dominant negative loss or alteration of VCP function culminating in impaired degradation of TDP-43. TDP-43 is a common pathologic substrate linking a variety of distinct patterns of FTLD-U pathology caused by different genetic alterations.

Seleno-L-methionine protects against beta-amyloid and iron/hydrogen peroxide-mediated neuron death.

Increasing evidence suggests a role for oxidative stress in several neurodegenerative diseases, including Alzheimer's disease (AD), and that selenium compounds may function as antioxidants. To evaluate the antioxidant mechanism of selenium, primary rat hippocampal neurons were pretreated with seleno-L-methionine (SeMet) for 16 h prior to treatment with iron/hydrogen peroxide (Fe(2+)/H(2)O(2)) or amyloid beta peptide (Abeta(2535)); free radical generation was assessed using laser confocal microscopy and CM-H(2)DCFDA and APF. Treatment with Fe(2+)/H(2)O(2) or Abeta significantly decreased cell survival and increased free radical generation compared to cultures treated with vehicle alone. In contrast, cultures pretreated with SeMet showed significantly (p < 0.05) increased survival and significantly lower CM-H(2)DCFDA and APF fluorescence compared to Fe(2+)/H(2)O(2) or Abeta treated cultures. To determine if SeMet protection was mediated by glutathione peroxidase (GPx), levels of GPx protein and activity were measured using confocal microscopy and a selenium-dependent GPx specific antibody and an activity assay. Pretreatment with SeMet significantly (p < 0.05) increased GPx protein and activity in Fe(2+)/H(2)O(2)- and Abeta-treated cultures compared to cultures treated with Fe(2+)/H(2)O(2) or Abeta alone. These data suggest that SeMet can decrease free radical generation induced by Fe(2+)/H(2)O(2) or Abeta through modulation of GPx and may be suitable as a potential therapeutic agent in neurodegenerative diseases where there is increased oxidative stress.

Novel ubiquitin neuropathology in frontotemporal dementia with valosin-containing protein gene mutations.

Frontotemporal dementia (FTD) with inclusion body myopathy and Paget disease of bone (IBMPFD) is a rare, autosomal-dominant disorder caused by mutations in the valosin-containing protein (VCP) gene, a member of the AAA-ATPase gene superfamily. The neuropathology associated with sporadic FTD is heterogeneous and includes tauopathies and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). However, there is limited information on the neuropathology in IBMPFD. We performed a detailed, systematic analysis of the neuropathologic changes in 8 patients with VCP mutations. A novel pattern of ubiquitin pathology was identified in IBMPFD that was distinct from sporadic and familial FTLD-U without VCP gene mutations. This was characterized by ubiquitin-positive neuronal intranuclear inclusions and dystrophic neurites. In contrast to FTLD-U, only rare intracytoplasmic inclusions were identified. The ubiquitin pathology was abundant in the neocortex, less robust in limbic and subcortical nuclei, and absent in the dentate gyrus. Only rare inclusions were detected with antibodies to VCP and there was no biochemical alteration in the VCP protein. VCP is associated with a variety of cellular activities, including regulation of the ubiquitin-proteasome system. Our findings are consistent with the hypothesis that the pathology associated with VCP gene mutations is the result of impairment of ubiquitin-based degradation pathways.

Elevated zinc transporter-6 in mild cognitive impairment, Alzheimer disease, and pick disease.

Filamentous cytoplasmic inclusions are hallmarks of Alzheimer disease (AD) and Pick disease (PD). Although previous studies show elevated zinc (Zn) in AD brain, there has been little study of zinc transporter (ZnT) proteins that are critical in the maintenance of Zn homeostasis. Using Western blot analysis, we show significantly elevated ZnT-6, the protein responsible for sequestration of Zn in the trans-Golgi network, in the hippocampus/parahippocampal gyrus (HPG) of AD subjects compared to age-matched controls and a trend toward elevated ZnT-6 in subjects with amnestic mild cognitive impairment (MCI). Based on these data, we used immunohistochemistry to investigate the cellular distribution of ZnT-6 in the HPG of control subjects and subjects with MCI, AD, and PD. Comparison of immediately adjacent serial sections stained using the modified Bielschowsky method and immunostained for ZnT-6 showed elevated ZnT-6 in 89 +/- 7% of neurofibrillary tangle (NFT)-bearing neurons in AD and 100 +/- 19% of Pick bodies in PD specimens. Confocal microscopy of HPG from MCI subjects double labeled for ZnT-6 and MC-1, a marker of early NFT formation, showed 85 +/- 4% of MC-1-positive cells were strongly ZnT-6-positive. Increased ZnT-6 immunostaining in neurons containing cytoplasmic inclusions in MCI, AD, and PD suggests a role for ZnT-6 in the pathogenesis of these lesions.

Proteasome inhibition induces reversible impairments in protein synthesis.

Proteasome inhibition occurs during normal aging and in a variety of age-related diseases, with inhibition of proteasome function sufficient to induce physiological and pathological alterations observed in each of these conditions. It is presumed that proteasome inhibition induces cellular alterations by promoting rapid protein accumulation, as the direct result of impairments in protein removal, which assumes protein synthesis remains relatively unchanged during proteasome inhibition. We conducted experimentation using established proteasome inhibitors and primary rat neuron cultures in order to elucidate whether proteasome inhibition had any effect on neuronal protein synthesis. Proteasome inhibition impaired neuronal protein synthesis, with concentrations of inhibitor necessary to significantly inhibit protein synthesis similar to the concentrations necessary to induce subsequent neuron death. The inhibition of protein synthesis was reversible during the first 6 h of treatment, with the neurotoxicity of proteasome inhibition reversible during the first 12 h of treatment. These studies are the first to demonstrate a potentially important interplay between the proteasome and protein synthesis in neurons, and the first to identify that some effects of proteasome inhibition are reversible in neurons. Together these findings have important implications for understanding proteasome inhibition as a potential contributor to aging and age-related disease.

Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease.

Mild cognitive impairment (MCI) is generally referred to the transitional zone between normal cognitive function and early dementia or clinically probable Alzheimer's disease (AD). Oxidative stress plays a significant role in AD and is increased in the superior/middle temporal gyri of MCI subjects. Because AD involves hippocampal-resident memory dysfunction, we determined protein oxidation and identified the oxidized proteins in the hippocampi of MCI subjects. We found that protein oxidation is significantly increased in the hippocampi of MCI subjects when compared to age- and sex-matched controls. By using redox proteomics, we determined the oxidatively modified proteins in MCI hippocampus to be alpha-enolase (ENO1), glutamine synthetase (GLUL), pyruvate kinase M2 (PKM2) and peptidyl-prolyl cis/trans isomerase 1 (PIN1). The interacteome of these proteins revealed that these proteins functionally interact with SRC, hypoxia-inducible factor 1, plasminogen (PLG), MYC, tissue plasminogen activator (PLAT) and BCL2L1. Moreover, the interacteome indicates the functional involvement of energy metabolism, synaptic plasticity and mitogenesis/proliferation. Therefore, oxidative inactivation of ENO1, GLUL and PIN1 may alter these cellular processes and lead to the development of AD from MCI. We conclude that protein oxidation plays a significant role in the development of AD from MCI and that the oxidative inactivation of ENO1, GLUL, PKM2 and PIN1 is involved in the progression of AD from MCI. The current study provides a framework for future studies on the development of AD from MCI relevant to oxidative stress.

Biophysical and biochemical characterization of the intrinsic fluorescence from neurofibrillary tangles.

Recently, we developed a novel fluorescent method named intrinsic fluorescence induction that allows direct visualization of neurofibrillary pathology without introducing exogenous chromogens. In the present study, we further characterized the properties of this novel red fluorescence biophysically, biochemically, and neuropathologically. In vitro spectrofluorometry and in situ emission scan show that the intrinsic fluorescence of neurofibrillary tangles has a long emission wavelength peak at 620 nm and a large Stoke's shift of 70 nm. Dephosphorylation of Alzheimer's disease brain sections with alkaline phosphatase or denaturation with guanidine only causes a subtle reduction in the induced fluorescence of neurofibrillary tangles, while hydrofluoric acid or formic acid completely eliminates the fluorescence. Chemical modification of residue serine, but not tyrosine or tryptophan, reduced the intensity of induced fluorescence significantly. The induced fluorophore, thus, has unique properties, and its generation likely depends on the particular conformation of paired helical filaments, which may in turn depend on tau hyperphosphorylation.

3alpha,5alpha-THP: a potential plasma neurosteroid biomarker in Alzheimer's disease and perhaps non-Alzheimer's dementia.

RATIONALE: A plasma biomarker for neurodegenerative disease is desirable because blood is relatively simple to obtain compared with other biological samples such as cerebrospinal fluid. Recent literature suggests that neurosteroid metabolism may be altered in Alzheimer's disease (AD). OBJECTIVES: We sought to measure the plasma levels of seven steroids to assess their potential as biomarkers for dementia and AD. METHODS: Steroids were measured using validated radioimmunoassay methods in AD (n=15), non-AD dementia (n=4), and control subjects (n=20). Demented subjects were in the mild-to-moderate stages of illness. Measurements were done blind to subject status in an independent laboratory. RESULTS: The notable finding was the significantly lower 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) level in demented subjects compared with controls (25% decrease; p=0.004); 3alpha,5alpha-THP was the only one of the steroids demonstrating an effect of dementia. CONCLUSION: Lowered 3alpha,5alpha-THP levels appear promising as a biomarker in dementia, but further work is needed to establish the sensitivity and specificity of these findings in AD.

Quantitative proteomic analysis of mitochondria from primary neuron cultures treated with amyloid beta peptide.

Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of neuron degeneration in Alzheimer's disease (AD). Although several studies have examined the effect of amyloid beta peptide (Abeta), on activities of individual proteins in primary neuron cultures, there have been no studies of the effects of Abeta on the mitochondrial proteome. Here, we quantitatively measured changes in mitochondrial proteins of primary rat cortical neuron cultures exposed to 25 microM Abeta(25-35) for 16 h using isotope coded affinity tag (ICAT) labeling and 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) which allows simultaneous identification and quantification of cysteine-containing proteins. The analysis of enriched mitochondrial fractions identified 10 proteins including sodium/potassium-transporting ATPase, cofilin, dihydropyrimidinase, pyruvate kinase and voltage dependent anion channel 1 that were statistically significantly (P < 0.05) altered in Abeta-treated cultures. Elevations of proteins associated with energy production suggest that cells undergoing Abeta-mediated apoptosis increase synthesis of proteins essential for ATP production and efflux in an attempt to maintain metabolic function.

Genetic and expression profiles of cerebellar liponeurocytomas.

Cerebellar liponeurocytoma, a rare, newly identified CNS neoplasm of adults, is characterized by advanced neuronal/neurocytic and focal lipomatous differentiation, low proliferative potential and a favorable clinical prognosis. Despite the different age distribution and benign biological behavior, the cerebellar liponeurocytoma shares several features with the cerebellar medulloblastoma, which may include an origin from the periventricular matrix of the fourth ventricle or the external granular layer of the cerebellum. To establish the genetic profile of cerebellar liponeurocytomas, we have formed an international consortium and collected tumor samples from 20 patients. DNA sequencing revealed TP53 missense mutations in 4 (20%) of 20 cerebellar liponeurocytomas, a frequency higher than in medulloblastomas. There was no case with PTCH, APC, or beta-catenin mutations, each of which may be present in subsets of medulloblastomas. Isochromosome 17q, a genetic hallmark of classic medulloblastomas, was not observed in any of the cases investigated by FISH analysis. cDNA array analyses were carried out on 4 cerebellar liponeurocytomas, 4 central neurocytomas, and 4 classic medulloblastomas. Cluster analysis of the cDNA expression data of 1176 genes grouped cerebellar liponeurocytomas close to central neurocytomas, but distinct from medulloblastomas. These results suggest cerebellar liponeurocytoma as a distinct tumor entity that is genetically different from medulloblastoma. Furthermore, the cDNA expression array data suggest a relationship to central neurocytomas, but the presence of TP53 mutations, which are absent in central neurocytomas, suggests that their genetic pathways are different.

4-Hydroxynonenal oxidatively modifies histones: implications for Alzheimer's disease.

There is increasing evidence of DNA oxidation and altered DNA repair mechanisms in Alzheimer's disease (AD) brain. Histones, which interact with DNA, conceivably could provide a protective shield for DNA against oxidative stress. However, because of their abundant lysine residues, histones may be a target for 4-hydroxynonenal (HNE) modification. In this study, we have shown that HNE binds to histones and that this binding affects the conformation of the histone, measured by electron paramagnetic resonance in conjunction with a protein-specific spin label. The covalent modification to the histone by HNE affects the ability of the histone to bind DNA. Interestingly, acetylated histones appear to be more susceptible to HNE modifications than control histones. Conceivably, altered DNA-histone interactions, subsequent to oxidative modification of histones by the lipid peroxidation product HNE, may contribute to the vulnerability of DNA to oxidation in AD brain.