Role of the Intestinal Microbiota in Host Responses to Stressor Exposure.

Humans have coevolved over time to not only tolerate but also rely on trillions of microbes that aid in the development of our immune system, provide nutrients, break down potentially noxious substances, and act as a barrier against potentially pathogenic organisms. These microbes, collectively known as the microbiota, live in relatively stable communities on mucosal surfaces such as the respiratory tract and gastrointestinal tract. Changes to the microbiota are often transient, due to changes in diet, antibiotic exposure, and psychological stressor exposure. This chapter will discuss how psychological stressors can shape the intestinal microbial community and how these perturbations can contribute to stressor-induced changes in immune function, neurodevelopment, and behavioral deficits.

Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia.

N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in excitatory neurotransmission, synaptic plasticity and brain development. Clinical and experimental evidence suggests a dysregulation of NMDAR function and glutamatergic pathways in the pathophysiology of schizophrenia. We evaluated electrophysiological and behavioral properties of NMDAR deficiency utilizing mice that express only 5-10% of the normal level of NMDAR NR1 subunit. Auditory and visual event related potentials yielded significantly increased amplitudes for the P20 and N40 components in NMDAR deficient (NR1(neo)-/-) mice suggesting decreased inhibitory tone. Compared to wild types, NR1(neo)-/- mice spent less time in social interactions and showed reduced nest building. NR1(neo)-/- mice displayed a preference for open arms of a zero maze and central zone of an open field, possibly reflecting decreased anxiety-related behavioral inhibition. However, locomotor activity did not differ between groups in either home cage environment or during behavioral testing. NR1(neo)-/- mice displayed hyperactivity only when placed in a large unfamiliar environment, suggesting that neither increased anxiety nor non-specific motor activation accounts for differential behavioral patterns. Data suggest that NMDAR NR1 deficiency causes disinhibition in sensory processing as well as reduced behavioral inhibition and impaired social interactions. The behavioral signature in NR1(neo)-/- mice supports the impact of impaired NMDAR function in a mouse model with possible relevance to negative symptoms in schizophrenia.

Effect of APOE genotype on microvascular basement membrane in Alzheimer's disease.

APOE4 homozygosity has been associated with an increased risk of sporadic Alzheimer's disease through a mechanism, which has yet to be defined. Recent evidence has suggested that microvascular basement membrane injury may be a critical factor in the pathogenesis of AD-related dementia. In previous studies, we have shown that the synaptic organizing protein agrin can be found in neurons, and is a major component of the brain microvascular basement membrane. Here, we compare the basement membrane surface area of cortical microvasculature in AD brains by staining with an anti-agrin antibody. Quantitative morphometric analysis was used to determine the mean basement area (micro(2)) of prefrontal cortical microvessels. An average of 10 capillaries was measured in each of 35 cases of AD genotyped for APOE status. APOE4,4 homozygotes had smaller capillary basement membrane areas (17.4 micro(2))+/-6.2) than APOE3,3 homozygotes (26.9 micro(2)+/-6.5), p<0.001. The capillary basement membrane areas (CBMA) of heterozygotes APOE3,4 did not differ significantly from APOE3,3 or APOE4,4. Braak stage did not contribute significantly to CBMA. However, a preliminary analysis suggests an interaction between APOE4,4 and Braak V-VI producing smaller CBMA, a finding which needs to be confirmed with a larger sample. These data support the hypothesis that APOE4,4 is associated with thinning of the microvascular basement membrane in Alzheimer's disease.

IBOX(2-(4'-dimethylaminophenyl)-6-iodobenzoxazole): a ligand for imaging amyloid plaques in the brain.

It is well known that overproduction and accumulation of beta-amyloid (Abeta) plaques in the brain is a key event in the pathogenesis of Alzheimer's disease (AD). Previously it was demonstrated that [125I]TZDM, 2-(4'-dimethylaminophenyl)-6-iodobenzothiazole, a thioflavin derivative, was an effective ligand with good in vitro and in vivo binding characteristics. To further improve the initial uptake and washout rate from the brain, important properties for in vivo imaging agents, a novel radioiodinated ligand, 2-(4'-dimethylaminophenyl)-6-iodobenzoxazole ([125I]IBOX, 3), for detecting Abeta plaques in the brain, was synthesized and evaluated. The new iodinated ligand, IBOX, is based on an isosteric replacement of a sulfur atom of TZDM by an oxygen, by which the molecular weight is reduced while the lipophilicity of the iodinated ligand is increased. Partition coefficients (P.C.) of these two ligands were 70 and 124 for TZDM and IBOX, respectively. In vitro binding study indicated that the isosteric displacement yielded a new ligand with equal binding potency to Abeta(1-40) aggregates (K(i) = 1.9 and 0.8 nM for TZDM and IBOX, respectively). Autoradiography of postmortem brain sections of a confirmed AD patient by [125I]IBOX showed excellent labeling of plaques similar to that observed with [125I]TZDM. More importantly, in vivo biodistribution of [125I]IBOX in normal mice displayed superior peak brain uptake (2.08% at 30 min vs 1.57% at 60 min dose/brain for [125I]IBOX and [125I]TZDM, respectively). In addition, the washout from the brain was much faster for [125I]IBOX as compared to [125I]TZDM. Based on the data presented for [125I]IBOX, it is predicted that the brain trapping of this new radioiodinated ligand in the Abeta containing regions will be more favorable than that of the parent compound, [125I]TZDM. Further evaluation of [125I]IBOX is warranted to confirm the Abeta plaque labeling properties in vivo.

Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates.

We report for the first time that small molecule-based radiodiodinated ligands, showing selective binding to Abeta aggregates, cross the intact blood-brain barrier by simple diffusion. Four novel ligands showing preferential labeling of amyloid aggregates of Abeta(1-40) and Abeta(1-42) peptides, commonly associated with plaques in the brain of people with Alzheimer's disease (AD), were developed. Two 125I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene, 13 (IMSB), and two 125I-labeled thioflavins, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM), and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands showed excellent binding affinities with Kd values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates of Abeta(1-40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Abeta(1-42), respectively. Interestingly, under a competitive-binding assaying condition, different binding sites on Abeta(1-40) and Abeta(1-42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down's syndrome known to contain primarily Abeta(1-42) aggregates in the brain showed that both [(125)I]18a and [125I]18b labeled these brain sections, but [125I]13, selective for Abeta(1-40) aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies in normal mice after an iv injection showed that [125I]18a and [(125)I]18b exhibited excellent brain uptake and retention, the levels of which were much higher than those of [125I]12 and [125I]13. These findings strongly suggest that the new radioiodinated ligands, [125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), may be useful as biomarkers for studying Abeta(1-40) as well as Abeta(1-42) aggregates of amyloidogenesis in AD patients.

Isomerization of (Z,Z) to (E,E)1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene in strong base: probes for amyloid plaques in the brain.

In developing probes for detecting beta-amyloid (Abeta) plaques in the brain of Alzheimer's disease (AD), we have synthesized 1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (5, BSB). Due to the presence of two double bonds, formation of four different isomers is possible. Four isomers, E,E-5, E,Z-5, Z,E-5, and Z,Z-5, were prepared. Surprisingly, all showed strong fluorescent labeling of Abeta plaques in the brain of postmortem brain sections of patients with confirmed AD. In vitro binding assay also showed that all four isomers of BSB (E,E-5, E,Z-5, Z,E-5, and Z,Z-5) displayed a similar high binding affinity inhibiting the binding of [(125)I]E,E-6, 1-iodo-2,5-bis-(3-hydroxycarbonyl-4-methoxy)styrylbenzene (IMSB) to Abeta(1-40) aggregates. The inhibition constants (K(i)) of E,E-5, E,Z-5, Z,E-5, and Z,Z-5 were 0.11 +/- 0.01, 0.19 +/- 0.03, 0.27 +/- 0.06, and 0.13 +/- 0.02 nM, respectively. Due to the fact that geometric stability of these styrylbenzenes is unknown, and the conversion of Z,Z-5 to E,E-5 may occur automatically in the binding or labeling assaying conditions, we have investigated the kinetics of conversion of Z,Z-5 to E,E-5 by NMR in D(2)O/NaOD at elevated temperatures (70, 95, and 115 degrees C). The activation energy was determined to be 14.15 kcal/mol. The results strongly suggest that the isomeric conversion at room temperature in aqueous buffer solution is unlikely. All of the styrylbenzene isomers clearly showed potential as useful tools for studying Abeta aggregates in the brain. The data suggest that, despite the rigidity of this series of styrylbenzenes, the binding sites on Abeta aggregates may have certain flexibility and the binding pockets could be adaptable for binding to other smaller ligands. Such information could be exploited to develop new ligands for detecting amyloid plaques in AD.

Widespread nitration of pathological inclusions in neurodegenerative synucleinopathies.

Reactive nitrogen species may play a mechanistic role in neurodegenerative diseases by posttranslationally altering normal brain proteins. In support of this hypothesis, we demonstrate that an anti-3-nitrotyrosine polyclonal antibody stains all of the major hallmark lesions of synucleinopathies including Lewy bodies, Lewy neurites and neuraxonal spheroids in dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and neurodegeneration with brain iron accumulation type 1, as well as glial and neuronal cytoplasmic inclusions in multiple system atrophy. This antibody predominantly recognized nitrated alpha-synuclein when compared to other in vitro nitrated constituents of these pathological lesions, such as neurofilament subunits and microtubules. Collectively, these findings imply that alpha-synuclein is nitrated in pathological lesions. The widespread presence of nitrated alpha-synuclein in diverse intracellular inclusions suggests that oxidation/nitration is involved in the onset and/or progression of neurodegenerative diseases.

Immunohistochemical and biochemical studies demonstrate a distinct profile of alpha-synuclein permutations in multiple system atrophy.

Although alpha-synuclein (alpha-syn) has been implicated as a major component of the abnormal filaments that form glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA), it is uncertain if GCIs are homogenous and contain full-length alpha-syn. Since this has implications for hypotheses about the pathogenesis of GCIs, we used a novel panel of antibodies to defined regions throughout alpha-syn in immunohistochemical epitope mapping studies of GCIs in MSA brains. Although the immunostaining profile of GCIs with these antibodies was similar for all MSA brains, there were significant differences in the immunoreactivity of the alpha-syn epitopes detected in GCIs. Notably, carboxy-terminal alpha-syn epitopes were immunodominant in GCIs, but the entire panel of antibodies immunostained cortical Lewy bodies (LBs) in dementia with LBs brain with similar intensity. While the distribution of alpha-syn labeled GCIs paralleled that previously reported using silver stains, antibodies to carboxy-terminal alpha-syn epitopes revealed a previously undescribed burden of GCIs in the MSA hippocampal formation. Finally, Western blots demonstrated detergent insoluble monomeric and high-molecular weight alpha-syn species in GCI rich MSA cerebellar white matter. Collectively, these data indicate that alpha-syn is a prominent component of GCIs in MSA, and that GCIs and LBs may result from cell type specific conformational or post-translational permutations in alpha-syn.

Biochemical alterations in neonatal hypoxic ischaemic brain damage.

Asphyxiated (n = 39) and control (n = 23) were elected for the study. Free radical-mediated lipid peroxidation, prostaglandin E2 and vitamin E levels were studied and the degree of hypoxic ischaemic encephalopathy was determined in each case. In the hypoxic group the concentration of prostaglandin E2 activity (P < 0.05) and malondialdehyde levels (P < 0.01) were significantly higher when compared to that of controls. The high vitamin E concentrations in the asphyxiated infants supports the role of oxygen free radicals in hypoxic ischaemic encephalopathy of newborns.

Methane synthesis on nickel by a solid-state ionic method.

The feasibility of electrochemically synthesizing methane by a Fischer-Tropsch type reaction by use of a solid oxide electrolyte has been demonstrated. This solid-state ionic approach provides in situ control of the oxygen activity at the gascatalyst interface by imposing a suitable voltage drop across an oxygen-conducting solid electrolyte from an external source. Methanation rates for hydrogen-carbon monoxide and hydrogen-carbon dioxide synthesis gas mixtures upon nickel electrodes showed substantial enhancement with the use of this technique, reaching values nearly two orders of magnitude higher than their intrinsic rates.