Comparative lipidomic analysis of mouse and human brain with Alzheimer disease

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.     

The unsolved relationship of brain aging and late-onset Alzheimer disease

Late-onset Alzheimer disease is the most common form of dementia and is strongly associated with age. Today, around 24 million people suffer from dementia and with aging of industrial populations this number will significantly increase throughout the next decades. An effective therapy that successfully decelerates or prevents the progressive neurodegeneration does not exist. Histopathologically Alzheimer disease is characterized by extensive extracellular amyloid β (Aβ) plaques, intracellular neurofibrillary tangles (NFTs), synaptic loss and neuronal cell death in distinct brain regions. The molecular correlation of Aβ or NFTs and development of late-onset Alzheimer disease needs further clarification. This review focuses on structural and functional alterations of the brain during aging, age-associated imbalances of defences against oxidative stress and age-related alterations of the metabolism of Aβ, via a comparison of observations in healthy aged individuals and cognitively impaired or AD patients. Although our understanding of brain region-specific neuronal aging is still incomplete, the early structural and molecular changes in the transition from cognitive health to impairment are subtle and the actual factors triggering the severe brain atrophy during LOAD remain ambiguous.     

Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer disease brain and in okadaic acid-treated SY5Y cells.

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation-dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF-H and NF-M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF-H and NF-M was 1.7- and 1.5-fold (P<0.01) as determined by monoclonal antibody SMI33, and was 1.6-fold (P<0.01) in NF-L using antibody NR4. The phosphorylation of NF-H and NF-M in AD was increased respectively at the SMI31 epitope by 1.6- and 1.9-fold (P<0.05) and at the SMI33 epitope by 2.7- and 1.3-fold (P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)-2A and -1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP-2A/PP-1 activities can lead to the hyperphosphorylation of NF-H and NF-M subunits.     

Targets of caspase-6 activity in human neurons and Alzheimer disease

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and alpha-Tubulin. A neoepitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.     

Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.     

Increased O-GlcNAc levels correlate with decreased O-GlcNAcase levels in Alzheimer disease brain

The potential role of the posttranslational modification of proteins with O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) in the pathogenesis of Alzheimer disease (AD) has been studied extensively, yet the exact function of O-GlcNAc in AD remains elusive. O-GlcNAc cycling is facilitated by only two highly conserved enzymes: O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase (OGA) catalyzes the removal of GlcNAc from proteins. Studies analyzing global O-GlcNAc levels in AD brain have produced inconsistent results and the reasons for altered O-GlcNAcylation in AD are still poorly understood. In this study, we show a 1.2-fold increase in cytosolic protein O-GlcNAc modification in AD brain when compared to age-matched controls. Interestingly, O-GlcNAc changes seem to be attributable to differential modification of a few individual proteins. While our finding of augmented O-GlcNAcylation concurs with some reports, it is contrary to others demonstrating decreased O-GlcNAc levels in AD brain. These conflicting results emphasize the need for further studies providing conclusive evidence on the subject of O-GlcNAcylation in AD. We further demonstrate that, while OGT protein levels are unaffected in AD, OGA protein levels are significantly decreased to 75% of those in control samples. In addition, augmented protein O-GlcNAc modification correlates to decreased OGA protein levels in AD subjects. While OGA inhibitors are already being tested for AD treatment, our results provide a strong indication that the general subject of O-GlcNAcylation and specifically its regulation by OGA and OGT in AD need further investigation to conclusively elucidate its potential role in AD pathogenesis and treatment.     

Increased levels of 4-hydroxynonenal and acrolein in the brain in preclinical Alzheimer disease

Previous studies demonstrate increased levels of 4-hydroxynonenal (HNE) and acrolein in vulnerable brain regions of subjects with mild cognitive impairment and late-stage Alzheimer disease (LAD). Recently preclinical AD (PCAD) subjects, who demonstrate normal antemortem neuropsychological test scores but abundant AD pathology at autopsy, have become the focus of increased study. Levels of extractable HNE and acrolein were quantified by gas chromatography–mass spectrometry with negative chemical ionization, and protein-bound HNE and acrolein were quantified by dot-blot immunohistochemistry in the hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyri (SMTG), and cerebellum (CER) of 10 PCAD and 10 age-matched normal control (NC) subjects. Results of the analyses show a significant (P < 0.05) increase in levels of extractable acrolein in the HPG of PCAD subjects compared to age-matched NC subjects and a significant decrease in extractable acrolein in PCAD CER. Significant increases in protein-bound HNE in HPG and a significant decrease in CER of PCAD subjects compared to NC subjects were observed. No significant alterations were observed in either extractable or protein-bound HNE or acrolein in the SMTG of PCAD subjects. Additionally, no significant differences in levels of protein carbonyls were observed in the HPG, SMTG, or CER of PCAD subjects compared to NC subjects.     

Down-regulation of cAMP-dependent protein kinase by over-activated calpain in Alzheimer disease brain

Impaired cognition and memory may be associated with down-regulation of cAMP-response element-binding protein (CREB) in the brain in patients with Alzheimer disease, but the molecular mechanism leading to the down-regulation is not understood. In this study, we found a selective reduction in the levels of the regulatory subunits (RIIα and RIIβ) and the catalytic subunit (Cβ) as well as the enzymatic activity of cAMP-dependent protein kinase (PKA), which is the major positive regulator of CREB. We also observed that PKA subunits were proteolyzed by calpain and the levels of PKA subunits correlated negatively with calpain activation in the human brain. These findings led us to propose that in the brain in patients with Alzheimer disease, over-activation of calpain because of calcium dysregulation causes increased degradation and thus decreased activity of PKA, which, in turn, contributes to down-regulation of CREB and impaired cognition and memory.     

Hyperphosphorylated tau in SY5Y cells: similarities and dissimilarities to abnormally hyperphosphorylated tau from Alzheimer disease brain.

Unlike normal tau, abnormally hyperphosphorylated tau (AD P-tau) from Alzheimer disease (AD) does not promote but instead inhibits microtubule assembly and disrupts already formed microtubules. Tau in the human neuroblastoma cell line SH-SY5Y is hyperphosphorylated at several of the same sites as AD P-tau, and accumulates in the cell body without any association to the cellular microtubule network. The aim of the present study was to elucidate why the SY5Y tau does not affect the viability of the cells. We found that, like AD P-tau, SY5Y tau because of hyperphosphorylation does not bind to microtubules and inhibits the tau-promoted assembly of microtubules. However, the tau/HMW MAP ratio is about 10 times less in SY5Y cells than in AD brain. These findings suggest that the hyperphosphorylated tau from SY5Y cells has similar biological characteristics as AD P-tau from AD brain, but is not lethal to the SY5Y cells because of its low tau/HMW MAP ratio.     

Linking variability in brain chemistry and circuit function through multimodal human neuroimaging

Identifying neurobiological mechanisms mediating the emergence of individual differences in behavior is critical for advancing our understanding of relative risk for psychopathology. Neuroreceptor positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) can be used to assay in vivo regional brain chemistry and function, respectively. Typically, these neuroimaging modalities are implemented independently despite the capacity for integrated data sets to offer unique insight into molecular mechanisms associated with brain function. Through examples from the serotonin and dopamine system and its effects on threat- and reward-related brain function, we review evidence for how such a multimodal neuroimaging strategy can be successfully implemented. Furthermore, we discuss how multimodal PET-fMRI can be integrated with techniques such as imaging genetics, pharmacological challenge paradigms and gene-environment interaction models to more completely map biological pathways mediating individual differences in behavior and related risk for psychopathology and inform the development of novel therapeutic targets.     

Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells

Iron- and aluminum-sulfate together, at nanomolar concentrations, trigger the production of reactive oxygen species (ROS) in cultures of human brain cells. Previous studies have shown that following ROS induction, a family of pathogenic brain genes that promote inflammatory signalling, cellular apoptosis and brain cell death is significantly over-expressed. Notably, iron- and aluminum-sulfate induce genes in cultured human brain cells that exhibit expression patterns similar to those observed to be up-regulated in moderate- to late-stage Alzheimer's disease (AD). In this study we have extended our investigations to analyze the expression of micro RNA (miRNA) populations in iron- and aluminum-sulfate treated human neural cells in primary culture. The main finding was that these ROS-generating neurotoxic metal sulfates also up-regulate a specific set of miRNAs that includes miR-9, miR-125b and miR-128. Notably, these same miRNAs are up-regulated in AD brain. These findings further support the idea that iron- and aluminum-sulfates induce genotoxicity via a ROS-mediated up-regulation of specific regulatory elements and pathogenic genes that redirect brain cell fate towards progressive dysfunction and apoptotic cell death.