Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer''s disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer''s disease patients'' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> aggravates vein graft intimal hyperplasia in a rat model

Background  

Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*10⁸ IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Cyclic 3'', 5''-AMP relay in dictyostelium discoideum. IV. Recovery of the cAMP signaling response after adaptation to cAMP

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.     

Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain

We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aβ-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aβ than ApoE4, but a greater proportion of Aβ molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aβ deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aβ and thereby increase the risk of AD for carriers of the APOEε4 allele.     

Monitoring protein aggregation and toxicity in Alzheimer's disease mouse models using in vivo imaging

Aggregation of amyloid beta peptide into senile plaques and hyperphosphorylated tau protein into neurofibrillary tangles in the brain are the pathological hallmarks of Alzheimer's disease. Despite over a century of research into these lesions, the exact relationship between pathology and neurotoxicity has yet to be fully elucidated. In order to study the formation of plaques and tangles and their effects on the brain, we have applied multiphoton in vivo imaging of transgenic mouse models of Alzheimer's disease. This technique allows longitudinal imaging of pathological aggregation of proteins and the subsequent changes in surrounding neuropil neurodegeneration and recovery after therapeutic interventions.     

Propagation of tau pathology in a model of early Alzheimer's disease

Neurofibrillary tangles advance from layer II of the entorhinal cortex (EC-II) toward limbic and association cortices as Alzheimer's disease evolves. However, the mechanism involved in this hierarchical pattern of disease progression is unknown. We describe a transgenic mouse model in which overexpression of human tau P301L is restricted to EC-II. Tau pathology progresses from EC transgene-expressing neurons to neurons without detectable transgene expression, first to EC neighboring cells, followed by propagation to neurons downstream in the synaptic circuit such as the dentate gyrus, CA fields of the hippocampus, and cingulate cortex. Human tau protein spreads to these regions and coaggregates with endogenous mouse tau. With age, synaptic degeneration occurs in the entorhinal target zone and EC neurons are lost. These data suggest that a sequence of progressive misfolding of tau proteins, circuit-based transfer to new cell populations, and deafferentation induced degeneration are part of a process of tau-induced neurodegeneration.     

Contributions of the hippocampus and medial prefrontal cortex to energy and body weight regulation

The effects of selective ibotenate lesions of the complete hippocampus (CHip), the hippocampal ventral pole (VP), or the medial prefrontal cortex (mPFC) in male rats were assessed on several measures related to energy regulation (i.e., body weight gain, food intake, body adiposity, metabolic activity, general behavioral activity, conditioned appetitive responding). The testing conditions were designed to minimize the nonspecific debilitating effects of these surgeries on intake and body weight. Rats with CHip and VP lesions exhibited significantly greater weight gain and food intake compared with controls. Furthermore, CHip-lesioned rats, but not rats with VP lesions, showed elevated metabolic activity, general activity in the dark phase of the light-dark cycle, and greater conditioned appetitive behavior, compared with control rats without these brain lesions. In contrast, rats with mPFC lesions were not different from controls on any of these measures. These results indicate that hippocampal damage interferes with energy and body weight regulation, perhaps by disrupting higher-order learning and memory processes that contribute to the control of appetitive and consummatory behavior.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background  

Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction.