Molecular abnormalities in autopsied brain tissue from the inferior horn of the lateral ventricles of nonagenarians and Alzheimer disease patients.

BACKGROUND: The ventricular system plays a vital role in blood-cerebrospinal fluid (CSF) exchange and interstitial fluid-CSF drainage pathways. CSF is formed in the specialized secretory tissue called the choroid plexus, which consists of epithelial cells, fenestrated capillaries and the highly vascularized stroma. Very little is currently known about the role played by the ventricles and the choroid plexus tissue in aging and Alzheimer's disease (AD). METHODS: In this study, we used our state-of-the-art proteomic platform, a liquid chromatography/mass spectrometry (LC-MS/MS) approach coupled with Tandem Mass Tag isobaric labeling to conduct a detailed unbiased proteomic analyses of autopsied tissue isolated from the walls of the inferior horn of the lateral ventricles in AD (77.2 ± 0.6 yrs), age-matched controls (77.0 ± 0.5 yrs), and nonagenarian cases (93.2 ± 1.1 yrs). RESULTS: Ingenuity pathway analyses identified phagosome maturation, impaired tight-junction signaling, and glucose/mannose metabolism as top significantly regulated pathways in controls vs nonagenarians. In matched-control vs AD cases we identified alterations in mitochondrial bioenergetics, oxidative stress, remodeling of epithelia adherens junction, macrophage recruitment and phagocytosis, and cytoskeletal dynamics. Nonagenarian vs AD cases demonstrated augmentation of oxidative stress, changes in gluconeogenesis-glycolysis pathways, and cellular effects of choroidal smooth muscle cell vasodilation. Amyloid plaque score uniquely correlated with remodeling of epithelial adherens junctions, Fc γ-receptor mediated phagocytosis, and alterations in RhoA signaling. Braak staging was uniquely correlated with altered iron homeostasis, superoxide radical degradation and phagosome maturation. CONCLUSIONS: These changes provide novel insights to explain the compromise to the physiological properties and function of the ventricles/choroid plexus system in nonagenarian aging and AD pathogenesis. The pathways identified could provide new targets for therapeutic strategies to mitigate the divergent path towards AD.

APOE genotype dependent molecular abnormalities in the cerebrovasculature of Alzheimer's disease and age-matched non-demented brains.

Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factors for AD is the apolipoprotein E4 (E4) allele. The APOE4 genotype has been shown to negatively impact vascular amyloid clearance, however, its direct influence on the molecular integrity of the cerebrovasculature compared to other APOE variants (APOE2 and APOE3) has been largely unexplored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains with different APOE genotypes. We first interrogated changes between healthy control cases to identify underlying genotype specific effects in cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. Oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction were the top significant pathways in E2E2 vs E3/E3cases. We also identified AD-dependent changes and their interactions with APOE genotype and found the highest number of significant proteins from this interaction was observed in the E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell-type specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E3/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E4/E4 cases. These proteomic changes provide novel insights into the longstanding link between APOE4 and cerebrovascular dysfunction, implicating a role for impaired autophagy, ER stress, and mitochondrial bioenergetics. These APOE4 dependent changes we identified could provide novel cerebrovascular targets for developing disease modifying strategies to mitigate the effects of APOE4 genotype on AD pathogenesis.

Molecular Pathobiology of the Cerebrovasculature in Aging and in Alzheimers Disease Cases With Cerebral Amyloid Angiopathy.

Cerebrovascular dysfunction and cerebral amyloid angiopathy (CAA) are hallmark features of Alzheimer's disease (AD). Molecular damage to cerebrovessels in AD may result in alterations in vascular clearance mechanisms leading to amyloid deposition around blood vessels and diminished neurovascular-coupling. The sequelae of molecular events leading to these early pathogenic changes remains elusive. To address this, we conducted a comprehensive in-depth molecular characterization of the proteomic changes in enriched cerebrovessel fractions isolated from the inferior frontal gyrus of autopsy AD cases with low (85.5 ± 2.9 yrs) vs. high (81 ± 4.4 yrs) CAA score, aged-matched control (87.4 ± 1.5 yrs) and young healthy control (47 ± 3.3 yrs) cases. We employed a 10-plex tandem isobaric mass tag approach in combination with our ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method. Enriched cerebrovascular fractions showed very high expression levels of proteins specific to endothelial cells, mural cells (pericytes and smooth muscle cells), and astrocytes. We observed 150 significantly regulated proteins in young vs. aged control cerebrovessels. The top pathways significantly modulated with aging included chemokine, reelin, HIF1α and synaptogenesis signaling pathways. There were 213 proteins significantly regulated in aged-matched control vs. high CAA cerebrovessels. The top three pathways significantly altered from this comparison were oxidative phosphorylation, Sirtuin signaling pathway and TCA cycle II. Comparison between low vs. high CAA cerebrovessels identified 84 significantly regulated proteins. Top three pathways significantly altered between low vs. high CAA cerebrovessels included TCA Cycle II, Oxidative phosphorylation and mitochondrial dysfunction. Notably, high CAA cases included more advanced AD pathology thus cerebrovascular effects may be driven by the severity of amyloid and Tangle pathology. These descriptive proteomic changes provide novel insights to explain the age-related and AD-related cerebrovascular changes contributing to AD pathogenesis. Particularly, disturbances in energy bioenergetics and mitochondrial biology rank among the top AD pathways altered in cerebrovessels. Targeting these failed mechanisms in endothelia and mural cells may provide novel disease modifying targets for developing therapeutic strategies against cerebrovascular deterioration and promoting cerebral perfusion in AD. Our future work will focus on interrogating and validating these novel targets and pathways and their functional significance.

The SUMO conjugation pathway in Populus: genomic analysis, tissue-specific and inducible SUMOylation and in vitro de-SUMOylation.

Covalent attachment of the small ubiquitin-like modifier (SUMO) to proteins in eukaryotic cells can regulate an assortment of cellular processes including transcription, and DNA-protein and protein-protein interactions. We identified gene models and found evidence for expression of genes involved in SUMOylation and SUMO deconjugation in Populus. We detected SUMOylated proteins in diverse organ and tissue types. SUMOylation was altered during responses to heat shock, desiccation, peroxide and irrigation of roots with high salt solution. SUMO deconjugation from substrates was sensitive to cysteine protease inhibitors. Product sizes and sensitivity to inhibitors are consistent with poly-SUMO chain formation as an intermediate step in SUMO redistribution to substrates in plant cells responding to treatments. The SUMOylation pathway is active in Populus and substrate conjugation to SUMO is a rapid response to multiple inducers.

Unbiased Proteomic Approach Identifies Unique and Coincidental Plasma Biomarkers in Repetitive mTBI and AD Pathogenesis.

The relationship between repetitive mild traumatic brain injury (r-mTBI) and Alzheimer's disease (AD) is well-recognized. However, the precise nature of how r-mTBI leads to or precipitates AD pathogenesis is currently not understood. Plasma biomarkers potentially provide non-invasive tools for detecting neurological changes in the brain, and can reveal overlaps between long-term consequences of r-mTBI and AD. In this study we address this by generating time-dependent molecular profiles of response to r-mTBI and AD pathogenesis in mouse models using unbiased proteomic analyses. To model AD, we used the well-validated hTau and PSAPP(APP/PS1) mouse models that develop age-related tau and amyloid pathological features, respectively, and our well-established model of r-mTBI in C57BL/6 mice. Plasma were collected at different ages (3, 9, and 15 months-old for hTau and PSAPP mice), encompassing pre-, peri- and post-"onset" of the cognitive and neuropathological phenotypes, or at different timepoints after r-mTBI (24 h, 3, 6, 9, and 12 months post-injury). Liquid chromatography/mass spectrometry (LC-MS) approaches coupled with Tandem Mass Tag labeling technology were applied to develop molecular profiles of protein species that were significantly differentially expressed as a consequence of mTBI or AD. Mixed model ANOVA after Benjamini-Hochberg correction, and a stringent cut-off identified 31 proteins significantly changing in r-mTBI groups over time and, when compared with changes over time in sham mice, 13 of these were unique to the injured mice. The canonical pathways predicted to be modulated by these changes were LXR/RXR activation, production of nitric oxide and reactive oxygen species and complement systems. We identified 18 proteins significantly changing in PSAPP mice and 19 proteins in hTau mice compared to their wild-type littermates with aging. Six proteins were found to be significantly regulated in all three models, i.e., r-mTBI, hTau, and PSAPP mice compared to their controls. The top canonical pathways coincidently changing in all three models were LXR/RXR activation, and production of nitric oxide and reactive oxygen species. This work suggests potential biomarkers for TBI and AD pathogenesis and for the overlap between these two, and warrant targeted investigation in human populations. Data are available via ProteomeXchange with identifier PXD010664.

Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine.

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Chronic elevation of phosphocholine containing lipids in mice exposed to Gulf War agents pyridostigmine bromide and permethrin.

For two decades, 25% of the veterans who served in the 1991 Gulf War (GW) have been living with Gulf War Illness (GWI), a chronic multisymptom illness. Evidence suggests that brain structures involved in cognitive function may be affected in GWI. Gulf War agents such as the acetylcholinesterase (AChE) inhibitor pyridostigmine bromide (PB) and the pesticide permethrin (PER) are considered key etiogenic factors in GWI. We therefore developed a mouse model of GW agent exposure by co-administering PB and PER and showed that this model exhibits cognitive impairment and anxiety, and increased astrogliosis at chronic post-exposure time-points. Since GW agents inhibit AChE, we hypothesized that PB+PER exposure will modulate phosphatidylcholine (PC) and sphingomyelin (SM), which are reservoirs of phosphocholine required for endogenous ACh synthesis. Lipidomic analyses showed that PC and SM were elevated in the brains of exposed compared to control mice. Brain ether PC (ePC) species were increased but lyso-platelet activating factors (lyso-PAF) that are products of ePC were decreased in exposed animals compared to controls. Catalase expression (a marker for peroxisomes) was increased in GW agent exposed mice compared to controls. Ether PC and lyso-PAF modulation was also evident in the plasma of GW agent exposed mice compared to controls. These studies suggest peroxisomal and lysosomal dysfunction in the brain at a chronic post-exposure timepoint following GW agent exposure. Our studies provide a new direction for GWI research, which will be useful for developing suitable therapies for treating GWI.

Lipidomic profiling of phosphocholine-containing brain lipids in mice with sensorimotor deficits and anxiety-like features after exposure to Gulf War agents.

The central nervous system (CNS)-based symptoms of Gulf War Illness (GWI) include motor dysfunction, anxiety, and cognitive impairment. Gulf War (GW) agents, such as pyridostigmine bromide (PB), permethrin (PER), N,N-diethyl-meta-toluamide (DEET), and stress, are among the contributory factors to the pathobiology of GWI. This study characterizes disturbances in phosphocholine-containing lipids that accompany neurobehavioral and neuropathological features associated with GW agent exposure. Exposed mice received PB orally, dermal application of PER and DEET and restraint stress daily for 28 days, while controls received vehicle during this period. Neurobehavioral studies included the rotarod, open field, and Morris water maze tests. Histopathological assessments included glial fibrillary acid protein, CD45, and Nissl staining. Liquid chromatography/mass spectrometry with source collision-induced dissociation in negative and positive ionization scanning modes was performed to characterize brain phosphatidylcholine (PC) and sphingomyelin (SM). A significant increase in ether containing PC (ePC34:0, ePC36:2, and ePC36:1) or long-chain fatty acid-containing PC (38:1, 40:4, 40:2) was observed in exposed mice compared with controls. Among differentially expressed PCs, levels of those with monounsaturated fatty acids were more affected than those with saturated and polyunsaturated fatty acids. Sensorimotor deficits and anxiety, together with an increase in astrocytosis, were observed in exposed mice compared with controls. These lipid changes suggest that alterations in peroxisomal pathways and stearoyl-CoA desaturase activity accompany neurobehavioral and neuropathological changes after GW agent exposure and represent possible treatment targets for the CNS symptoms of GWI.