Myles Mullan

Inhibition of angiogenesis by Abeta peptides.

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.

Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells

By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48h after a 30min cocaine exposure compared to control cells, and that this was followed by cell death at 72h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain.

APOLIPOPROTEIN E-GENOTYPE DEPENDENT HIPPOCAMPAL AND CORTICAL RESPONSES TO TRAUMATIC BRAIN INJURY

The different alleles of the apolipoprotein E gene (APOE-gene, ApoE-protein) have been reported to influence recovery after traumatic brain injury (TBI) in both human patients and animal models, with the e4 allele typically conferring poorer prognosis for recovery. How the E4 allele, and consequently the ApoE4 isoform, affects recovery is unknown, but proposed mechanisms include neurogenesis, inflammatory response and amyloid processing or metabolism. Using the controlled cortical impact (CCI) model of brain injury and microarray technology we have characterized the genomic response to injury in the brains of APOE2, APOE3 and APOE4 transgenic mice and identified quantitatively and qualitatively significantly different profiles of gene expression in both the hippocampus and the cortex of the APOE3 mice compared to APOE4. The observed gene regulation predicts functional consequences including effects on inflammatory processes, cell growth and proliferation, and cellular signaling, and may suggest that the poor recovery post-TBI in APOE4 animals and human patients is less likely to result from a specific activation of neurodegenerative mechanisms than a loss of reparative capability.

Apolipoprotein E genotype and oxidative stress response to traumatic brain injury.

Traumatic Brain Injury (TBI) is known to result in oxidative stress, and as variation at the Apolipoprotein E (APOE) gene has been shown to influence outcome following TBI, but through as yet unclear mechanisms, we used transgenic APOE mouse models to examine the relationship between APOE genotype and oxidative stress following TBI. We administered a controlled cortical impact (CCI) injury or sham injury to transgenic mice expressing either human APOE3 or APOE4 on a murine APOE-deficient background. RNA was prepared from the ipsilateral hippocampi and cortices retrieved at 24 h and 1 month post-TBI. Microarray analysis was performed on unpooled samples from three mice per group to determine the genomic response to TBI and to specifically investigate the response of genes involved in oxidative stress mechanisms. Our data demonstrated TBI-induced expression of many more anti-oxidant related genes in the APOE3 mice, suggesting a potential anti-oxidative role for ApoE3 compared to ApoE4. However, in an additional cohort of mice we isolated the ipsilateral hippocampi, cortices, and cerebella at 1 month after TBI or sham injury for immunohistochemical analysis of markers of oxidative stress: the formation and presence of carbonyls (indication of general oxidative modification), 3-nitrotyrosine (3NT; specific to protein modification), or 4-hydroxyl-2-nonenal (HNE; specific to lipid peroxidation). Although we observed significant increases in all three markers of oxidative stress in response to injury, and genotype was a significant factor for carbonyl and 3NT, we found no significant interaction between genotype and injury. This may be due to the overwhelming effect of injury compared to genotype in our ANOVA, but nonetheless suggests that an influence on oxidative stress response is not the primary mechanism behind the APOE-genotype dependent effects on outcome following TBI.

Diagnostic utility of APOE, soluble CD40, CD40L, and Aβ1–40 levels in plasma in Alzheimer’s disease

A continuous inflammatory state is associated with Alzheimers disease (AD) evidenced by an increase in proinflammatory cytokines around beta-amyloid (Abeta) deposits. In addition, functional loss of CD40L is shown to result in diminished Amyloid precursor proton (APP) processing and microglial activation, supporting a prominent role of CD40-CD40L in AD etiology. We therefore hypothesize that a peripheral increase in Abeta may result in corresponding increase of sCD40 and sCD40L further contributing to AD pathogenesis. We measured plasma Abeta, sCD40 and sCD40L levels in 73 AD patients and compared to 102 controls matched on general demographics. We demonstrated that Abeta(1-40), levels of sCD40 and sCD40L are increased in AD and declining MMSE scores correlated with increasing sCD40L, which in turn, correlated positively with Abeta(1-42). We then combined sCD40, sCD40L, Abeta and APOE and found that this biomarker panel has high sensitivity and specificity (>90%) as a predictor of clinical AD diagnosis. Given the imminent availability of potentially disease modifying therapies for AD, a great need exists for peripheral diagnostic markers of AD. Thus, we present preliminary evidence for potential usefulness for combination of plasma sCD40, sCD40L along with Abeta(1-40) and APOE epsilon4 in improving the clinical diagnosis of AD.

Amelioration of Experimental Autoimmune Encephalomyelitis by Anatabine

Anatabine, a naturally occurring alkaloid, is becoming a commonly used human food supplement, taken for its claimed anti-inflammatory properties although this has not yet been reported in human clinical trials. We have previously shown that anatabine does display certain anti-inflammatory properties and readily crosses the blood-brain barrier suggesting it could represent an important compound for mitigating neuro-inflammatory conditions. The present study was designed to determine whether anatabine had beneficial effects on the development of experimental autoimmune encephalomyelitis (EAE) in mice and to precisely determine its underlying mechanism of action in this mouse model of multiple sclerosis (MS). We found that orally administered anatabine markedly suppressed neurological deficits associated with EAE. Analyses of cytokine production in the periphery of the animals revealed that anatabine significantly reduced Th1 and Th17 cytokines known to contribute to the development of EAE. Anatabine appears to significantly suppress STAT3 and p65 NFκB phosphorylation in the spleen and the brain of EAE mice. These two transcription factors regulate a large array of inflammatory genes including cytokines suggesting a mechanism by which anatabine antagonizes pro-inflammatory cytokine production. Additionally, we found that anatabine alleviated the infiltration of macrophages/microglia and astrogliosis and significantly prevented demyelination in the spinal cord of EAE mice. Altogether our data suggest that anatabine may be effective in the treatment of MS and should be piloted in clinical trials.

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.

High serum Abeta and vascular risk factors in first-degree relatives of Alzheimer's disease patients.

The main objective of this study was to determine whether elevated blood β-amyloid (Aβ) levels among the first-degree relatives of patients with Alzheimer’s Disease (AD) are associated with vascular risk factors of AD. Serum Aβ was measured in samples from 197 cognitively normal first-degree relatives of patients with AD-like dementia. Study participants were recruited as part of an ancillary study of the Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT subpopulation). The ADAPT subpopulation was found to be similar in age, sex, and ethnicity to another cognitively normal cohort (n = 98). Using cross-sectional analyses, we examined the association of Aβ with blood pressure, lipid levels, apolipoprotein E genotypes, and the use of prescribed medication to treat vascular risk factors in the ADAPT subpopulation. Aβ1–40 was positively associated with age, use of antihypertensives, and serum creatinine, and we observed a marginal negative interaction on Aβ1–40 associated with systolic blood pressure and use of antihypertensives. Serum Aβ1–42 was associated with statin use and a positive correlation of Aβ1–42 with HDL was observed among statin nonusers. These findings suggest that high Aβ in the periphery among the family history-enriched cohorts may be due to enrichment of vascular risk factors and may reflect presymptomatic AD pathology. It remains to be determined whether the association of Aβ with medications used for treating vascular risk factors indicates prevention of AD. Longitudinal evaluation of blood Aβ in this cohort will provide a better understanding of the significance of this association in AD etiology.

Impaired Orthotopic Glioma Growth and Vascularization in Transgenic Mouse Models of Alzheimer's Disease

Alzheimers disease (AD) is the most common form of dementia among the aging population and is characterized pathologically by the progressive intracerebral accumulation of β-amyloid (Aβ) peptides and neurofibrillary tangles. The level of proangiogenic growth factors and inflammatory mediators with proangiogenic activity is known to be elevated in AD brains which has led to the supposition that the cerebrovasculature of AD patients is in a proangiogenic state. However, angiogenesis depends on the balance between proangiogenic and antiangiogenic factors and the brains of AD patients also show an accumulation of endostatin and Aβ peptides which have been shown to be antiangiogenic. To determine whether angiogenesis is compromised in the brains of two transgenic mouse models of AD overproducing Aβ peptides (Tg APPsw and Tg PS1/APPsw mice), we assessed the growth and vascularization of orthotopically implanted murine gliomas since they require a high degree of angiogenesis to sustain their growth. Our data reveal that intracranial tumor growth and angiogenesis is significantly reduced in Tg APPsw and Tg PS1/APPsw mice compared with their wild-type littermates. In addition, we show that Aβ inhibits the angiogenesis stimulated by glioma cells when cocultured with human brain microvascular cells on a Matrigel layer. Altogether our data suggest that the brain of transgenic mouse models of AD does not constitute a favorable environment to support neoangiogenesis and may explain why vascular insults synergistically precipitate the cognitive presentation of AD.

Serum β-Amyloid Correlates with Neuropsychological Impairment

ABSTRACT Aims: Evidence suggests a relationship between peripheral Aβ and AD. We hypothesized that higher levels of serum Aβ1–42 would be associated with memory impairment, thought to occur early in the disease, and rises in serum Aβ1–40, which occur later, would be associated with impairment in non-memory measures. Methods: Using a cross-sectional design, we examined the relationship of serum Aβ1–40, Aβ1–42, and the ratio of Aβ1–42/1–40 to neuropsychological measures in 40 cognitively normal controls, 13 MCI subjects, and 25 AD patients. Results: Serum Aβ1–42 and the ratio of Aβ1–42/1–40 were significantly higher in the MCI group compared to the controls. A significant relationship in the hypothesized direction (poorer scores associated with higher Aβ1–40 serum levels) was found between Aβ1–40 and measures of executive functions across the entire cohort of individuals tested and with measures of language and processing speed in the AD group. Regression analysis found that neuropsychological measures accounted for 26% of the variance in serum Aβ1–40, in the MCI/AD but not the controls. Furthermore that language and executive measures were significant predictors. Conclusions: Results provide preliminary data to partially support our hypotheses and suggest that changes in serum Aβ levels may be attributed to pathological changes within the brain.