Jon Reed

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.

Anatabine lowers Alzheimer's Aβ production in vitro and in vivo.

Brain Aβ accumulation represents a key pathological hallmark in Alzheimers disease. In this study, we investigated the impact of anatabine, a minor alkaloid present in plants of the Solanacea family on Aβ production in vitro using a cell line overexpressing the human amyloid precursor protein (APP) and in vivo using a transgenic mouse model of Alzheimers disease. In vitro, anatabine lowers Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a dose dependent manner and reduces sAPPβ production without impacting sAPPα levels suggesting that anatabine lowers Aβ production by mainly impacting the β-cleavage of APP. Additionally, we show that anatabine lowers NFκB activation at doses that inhibit Aβ production in vitro. Since NFκB is known to regulate BACE-1 expression (the rate limiting enzyme responsible for Aβ production), we determined the impact of anatabine on BACE-1 transcription. We show that anatabine inhibits BACE-1 transcription and reduces BACE-1 protein levels in human neuronal like SHSY-5Y cells suggesting that the Aβ lowering properties of anatabine are mediated via a regulation of BACE-1 expression. In vivo, we show that an acute treatment with anatabine for four days significantly lowers brain soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a transgenic mouse model of Alzheimers disease. Altogether our data suggest that anatabine may represent an interesting compound for regulating brain Aβ accumulation.

Exposure to an organophosphate pesticide, individually or in combination with other Gulf War agents, impairs synaptic integrity and neuronal differentiation, and is accompanied by subtle microvascular injury in a mouse model of Gulf War agent exposure.

Gulf War illness (GWI) is a currently untreatable multi‐symptom disorder experienced by 1990–1991 Persian Gulf War (GW) veterans. The characteristic hallmarks of GWI include cognitive dysfunction, tremors, migraine, and psychological disturbances such as depression and anxiety. Meta‐analyses of epidemiological studies have consistently linked these symptomatic profiles to the combined exposure of GW agents such as organophosphate‐based and pyrethroid‐based pesticides (e.g. chlorpyrifos (CPF) and permethrin (PER) respectively) and the prophylactic use of pyridostigmine bromide (PB) as a treatment against neurotoxins. Due to the multi‐symptomatic presentation of this illness and the lack of available autopsy tissue from GWI patients, very little is currently known about the distinct early pathological profile implicated in GWI (including its influence on synaptic function and aspects of neurogenesis). In this study, we used preclinical models of GW agent exposure to investigate whether 6‐month‐old mice exposed to CPF alone, or a combined dose of CPF, PB and PER daily for 10 days, demonstrate any notable pathological changes in hippocampal, cortical (motor, piriform) or amygdalar morphometry. We report that at an acute post‐exposure time point (after 3 days), both exposures resulted in the impairment of synaptic integrity (reducing synaptophysin levels) in the CA3 hippocampal region and altered neuronal differentiation in the dentate gyrus (DG), demonstrated by a significant reduction in doublecortin positive cells. Both exposures also significantly increased astrocytic GFAP immunoreactivity in the piriform cortex, motor cortex and the basolateral amygdala and this was accompanied by an increase in (basal) brain acetylcholine (ACh) levels. There was no evidence of microglial activation or structural deterioration of principal neurons in these regions following exposure to CPF alone or in combination with PB and PER. Evidence of subtle microvascular injury was demonstrated by the reduction of platelet endothelial cell adhesion molecule (PECAM)‐1 levels in CPF+PB+PER exposed group compared to control. These data support early (subtle) neurotoxic effects on the brain following exposure to GW agents.

Lipidomic analyses identify injury-specific phospholipid changes 3 mo after traumatic brain injury

Phospholipid (PL) abnormalities are observed in the cerebrospinal fluid of patients with traumatic brain injury (TBI), suggesting their role in TBI pathology. Therefore, PL levels were examined in a TBI mouse model that received 1.8 mm deep controlled cortical impact injury or craniectomy only (control). The rotarod and Barnes maze acquisition and probe tests were performed within 2 wk after injury, with another probe test performed 3 mo postinjury. Liquid chromatography/mass spectrometry analyses were performed on lipid extracts from several brain regions and plasma from injured and control mice collected at 3 mo postinjury. Compared to controls, injured mice with sensorimotor and learning deficits had decreased levels of cortical and cerebellar phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels, while hippocampal PC, sphingomyelin and PE levels were elevated. Ether PE levels were lower in the cortices and plasma of injured animals. Polyunsaturated fatty acid‐containing PC and PE species, particularly ratios of docosahexaenoic acid (DHA) to arachidonic acid, were lower in the hippocampi and cortices and plasma of injured mice. Given the importance of DHA in maintaining neuronal function and resolving inflammation and of peroxisomes in synthesis of ether PLs, normalizing these PLs may be a useful strategy for treating the chronic pathology of TBI.—Abdullah, L., Evans, J. E., Ferguson, S., Mouzon, B., Montague, H., Reed, J., Crynen, G., Emmerich, T., Crocker, M., Pelot, R., Mullan, M., Crawford, F., Lipidomic analyses identify injury‐specific phospholipid changes 3 months after traumatic brain injury. FASEB J. 28, 5311–5321 (2014). www.fasebj.org

Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation

Previous investigations have demonstrated the anti-inflammatory effects of cholinergic agonists, such as nicotine. In the present study, we investigated the potential anti-inflammatory activity of anatabine, a minor tobacco alkaloid also present in plants of the Solanacea family which displays a chemical structural similarity with nicotine. Our data show that anatabine prevents STAT3 and NFκB phosphorylation induced by lipopolysaccharide (LPS) or TNF-α in SH-SY5Y, HEK293, human microglia and human blood mononuclear cells. Using human whole blood, we found that anatabine prevents IL-1β production induced by LPS. We assessed anatabines anti-inflammatory activity in vivo using an acute model of inflammation by challenging wild-type mice with LPS. We observed that anatabine reduces pro-inflammatory cytokine production (IL-6, IL-1β and TNF-α) in the plasma, kidney and spleen of the animals following the injection of LPS and concomitantly opposes STAT3 phosphorylation induced by LPS in the spleen and kidney. We also investigated the impact of anatabine on neuroinflammation using a transgenic mouse model of Alzheimers disease (Tg APPsw) that displays elevated cytokine levels in the brain. Following a chronic oral treatment with anatabine, a reduction in brain TNF-α and IL-6 levels compared to untreated Tg APPsw mice was observed. Moreover, an increased STAT3 phosphorylation was detected in the brains of Tg APPsw mice compared to wild-type littermates and was inhibited by anatabine treatment. Overall our data show that the anti-inflammatory activity of anatabine in vitro and in vivo is mediated in part via an inhibition of STAT3 phosphorylation.

Identification of Plasma Biomarkers of TBI Outcome Using Proteomic Approaches in an APOE Mouse Model

The current lack of diagnostic and prognostic biomarkers for traumatic brain injury (TBI) confounds treatment and management of patients and is of increasing concern as the TBI population grows. We have generated plasma proteomic profiles from mice receiving TBI by controlled cortical impact at either 1.3 mm or 1.8 mm depth, comparing these against those of sham injured-animals to identify plasma biomarkers specific to mild or severe TBI at 24 hours, 1 month, or 3 months post-injury. To identify possible prognostic biomarkers, we used apolipoprotein E (APOE)3 and APOE4 transgenic mice, which demonstrate relatively favorable and unfavorable outcomes respectively, following TBI. Using a quantitative proteomics approach (isobaric tagging for relative and absolute quantitation--iTRAQ) we have identified proteins that are significantly modulated as a function of TBI and also in response to the TBI*APOE genotype interaction, the latter representing potential prognostic biomarkers. These preliminary data clearly demonstrate plasma protein changes that are not only injury dependent but also interaction dependent. Importantly, these results demonstrate the presence of TBI-dependent and interaction-dependent plasma proteins at a 3-month time point, which is a considerable time post-injury in the mouse model, and will potentially be of significance for combat veterans receiving assessment at extended periods post-injury. Furthermore, our identification of clusters of functionally related proteins indicates disturbance of particular biological modules, which potentially increases their value beyond that of solitary biomarkers.

Depletion of CXCR2 inhibits γ-secretase activity and amyloid-β production in a murine model of Alzheimer’s disease

Alzheimers disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aβ) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aβ levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aβ with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aβ promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.

Gulf War agent exposure causes impairment of long-term memory formation and neuropathological changes in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component such as memory deficits, neurological, and musculoskeletal problems. There are ample data that demonstrate that exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and pesticides such as permethrin (PER), were key contributors to the etiology of GWI post deployment to the Persian GW. In the current study, we examined the consequences of acute (10 days) exposure to PB and PER in C57BL6 mice. Learning and memory tests were performed at 18 days and at 5 months post-exposure. We investigated the relationship between the cognitive phenotype and neuropathological changes at short and long-term time points post-exposure. No cognitive deficits were observed at the short-term time point, and only minor neuropathological changes were detected. However, cognitive deficits emerged at the later time point and were associated with increased astrogliosis and reduction of synaptophysin staining in the hippocampi and cerebral cortices of exposed mice, 5 months post exposure. In summary, our findings in this mouse model of GW agent exposure are consistent with some GWI symptom manifestations, including delayed onset of symptoms and CNS disturbances observed in GWI veterans.

Effect of Venlafaxine and Desvenlafaxine on Drug Efflux Protein Expression and Biodistribution In Vivo

Venlafaxine, and to a lesser extent desvenlafaxine, has previously been shown to induce the expression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in whole cells and alter the cellular permeability of a known drug efflux probe (rhodamine 123). To validate these in vitro findings, wild-type mice were treated for 4 days with 10 mg/kg venlafaxine or desvenlafaxine, and drug efflux transporter expression was examined in the brain, liver, and intestine. P-gp and BCRP expression was significantly upregulated in the intestine, following a treatment with venlafaxine (2.6- and 6.7-fold, respectively) or desvenlafaxine (2.3- and 4.8-fold, respectively). In addition, venlafaxine increased the BCRP expression in the brain (40%) and liver (60%), whereas desvenlafaxine had no effect on drug efflux transporter levels in these tissues. Using the same treatment paradigm, we observed a minimal impact of either drug on the brain disposition of the known drug efflux probe, topotecan. However, in the periphery, venlafaxine treatment significantly reduced the topotecan oral bioavailability by nearly 40%, whereas the impact of desvenlafaxine on topotecan plasma levels was more modest (23%). These studies demonstrate an effect of venlafaxine on the drug efflux transport activity and the potential for clinical drug-drug interactions.

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine.

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.