Kevin X. Le

Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline

The complement system is part of the innate immune response responsible for removing pathogens and cellular debris, in addition to helping to refine CNS neuronal connections via microglia-mediated pruning of inappropriate synapses during brain development. However, less is known about the role of complement during normal aging. Here, we studied the role of the central complement component, C3, in synaptic health and aging. We examined behavior as well as electrophysiological, synaptic, and neuronal changes in the brains of C3-deficient male mice (C3 KO) compared with age-, strain-, and gender-matched C57BL/6J (wild-type, WT) control mice at postnatal day 30, 4 months, and 16 months of age. We found the following: (1) region-specific and age-dependent synapse loss in aged WT mice that was not observed in C3 KO mice; (2) age-dependent neuron loss in hippocampal CA3 (but not in CA1) that followed synapse loss in aged WT mice, neither of which were observed in aged C3 KO mice; and (3) significantly enhanced LTP and cognition and less anxiety in aged C3 KO mice compared with aged WT mice. Importantly, CA3 synaptic puncta were similar between WT and C3 KO mice at P30. Together, our results suggest a novel and prominent role for complement protein C3 in mediating aged-related and region-specific changes in synaptic function and plasticity in the aging brain. SIGNIFICANCE STATEMENT The complement cascade, part of the innate immune response to remove pathogens, also plays a role in synaptic refinement during brain development by the removal of weak synapses. We investigated whether complement C3, a central component, affects synapse loss during aging. Wild-type (WT) and C3 knock-out (C3 KO) mice were examined at different ages. The mice were similar at 1 month of age. However, with aging, WT mice lost synapses in specific brain regions, especially in hippocampus, an area important for memory, whereas C3 KO mice were protected. Aged C3 KO mice also performed better on learning and memory tests than aged WT mice. Our results suggest that complement C3, or its downstream signaling, is detrimental to synapses during aging.

Pyroglutamate-3 Amyloid-β Deposition in the Brains of Humans, Non-Human Primates, Canines, and Alzheimer Disease–Like Transgenic Mouse Models

Amyloid-β (Aβ) peptides, starting with pyroglutamate at the third residue (pyroGlu-3 Aβ), are a major species deposited in the brain of Alzheimer disease (AD) patients. Recent studies suggest that this isoform shows higher toxicity and amyloidogenecity when compared to full-length Aβ peptides. Here, we report the first comprehensive and comparative IHC evaluation of pyroGlu-3 Aβ deposition in humans and animal models. PyroGlu-3 Aβ immunoreactivity (IR) is abundant in plaques and cerebral amyloid angiopathy of AD and Down syndrome patients, colocalizing with general Aβ IR. PyroGlu-3 Aβ is further present in two nontransgenic mammalian models of cerebral amyloidosis, Caribbean vervets, and beagle canines. In addition, pyroGlu-3 Aβ deposition was analyzed in 12 different AD-like transgenic mouse models. In contrast to humans, all transgenic models showed general Aβ deposition preceding pyroGlu-3 Aβ deposition. The findings varied greatly among the mouse models concerning age of onset and cortical brain region. In summary, pyroGlu-3 Aβ is a major species of β-amyloid deposited early in diffuse and focal plaques and cerebral amyloid angiopathy in humans and nonhuman primates, whereas it is deposited later in a subset of focal and vascular amyloid in AD-like transgenic mouse models. Given the proposed decisive role of pyroGlu-3 Aβ peptides for the development of human AD pathology, this study provides insights into the usage of animal models in AD studies.

Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice

C3 deficiency protects against hippocampal neurodegeneration and cognitive decline in aged APP/PS1 mice despite abundant Aβ plaques. Avoiding complements Complement C3 is an immune molecule that protects against pathogens and plays a role in refinement of the developing visual system by removing weak nerve connections (that is, synapses). C3 is up-regulated in Alzheimer’s disease and, therefore, may contribute to the synapse loss that underlies cognitive decline. Shi et al. now report that an aged transgenic mouse model of Alzheimer’s disease that lacks C3 was protected against synapse loss and cognitive decline even in the presence of Aβ plaques, possibly by altering the glial response to Aβ deposition. Thus, modulation of complement signaling may have potential as a new therapeutic strategy for Alzheimer’s disease. The complement cascade not only is an innate immune response that enables removal of pathogens but also plays an important role in microglia-mediated synaptic refinement during brain development. Complement C3 is elevated in Alzheimer’s disease (AD), colocalizing with neuritic plaques, and appears to contribute to clearance of Aβ by microglia in the brain. Previously, we reported that C3-deficient C57BL/6 mice were protected against age-related and region-specific loss of hippocampal synapses and cognitive decline during normal aging. Furthermore, blocking complement and downstream iC3b/CR3 signaling rescued synapses from Aβ-induced loss in young AD mice before amyloid plaques had accumulated. We assessed the effects of C3 deficiency in aged, plaque-rich APPswe/PS1dE9 transgenic mice (APP/PS1;C3 KO). We examined the effects of C3 deficiency on cognition, Aβ plaque deposition, and plaque-related neuropathology at later AD stages in these mice. We found that 16-month-old APP/PS1;C3 KO mice performed better on a learning and memory task than did APP/PS1 mice, despite having more cerebral Aβ plaques. Aged APP/PS1;C3 KO mice also had fewer microglia and astrocytes localized within the center of hippocampal Aβ plaques compared to APP/PS1 mice. Several proinflammatory cytokines in the brain were reduced in APP/PS1;C3 KO mice, consistent with an altered microglial phenotype. C3 deficiency also protected APP/PS1 mice against age-dependent loss of synapses and neurons. Our study suggests that complement C3 or downstream complement activation fragments may play an important role in Aβ plaque pathology, glial responses to plaques, and neuronal dysfunction in the brains of APP/PS1 mice.

In Vivo Detection of Age- and Disease-Related Increases in Neuroinflammation by 18F-GE180 TSPO MicroPET Imaging in Wild-Type and Alzheimer's Transgenic Mice

Alzheimers disease (AD) is the most common cause of dementia. Neuroinflammation appears to play an important role in AD pathogenesis. Ligands of the 18 kDa translocator protein (TSPO), a marker for activated microglia, have been used as positron emission tomography (PET) tracers to reflect neuroinflammation in humans and mouse models. Here, we used the novel TSPO-targeted PET tracer 18F-GE180 (flutriciclamide) to investigate differences in neuroinflammation between young and old WT and APP/PS1dE9 transgenic (Tg) mice. In vivo PET scans revealed an overt age-dependent elevation in whole-brain uptake of 18F-GE180 in both WT and Tg mice, and a significant increase in whole-brain uptake of 18F-GE180 (peak-uptake and retention) in old Tg mice compared with young Tg mice and all WT mice. Similarly, the 18F-GE180 binding potential in hippocampus was highest to lowest in old Tg > old WT > young Tg > young WT mice using MRI coregistration. Ex vivo PET and autoradiography analysis further confirmed our in vivo PET results: enhanced uptake and specific binding (SUV75%) of 18F-GE180 in hippocampus and cortex was highest in old Tg mice followed by old WT, young Tg, and finally young WT mice. 18F-GE180 specificity was confirmed by an in vivo cold tracer competition study. We also examined 18F-GE180 metabolites in 4-month-old WT mice and found that, although total radioactivity declined over 2 h, of the remaining radioactivity, ∼90% was due to parent 18F-GE180. In conclusion, 18F-GE180 PET scans may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment. SIGNIFICANCE STATEMENT Microglial activation, a player in Alzheimers disease (AD) pathogenesis, is thought to reflect neuroinflammation. Using in vivo microPET imaging with a novel TSPO radioligand, 18F-GE180, we detected significantly enhanced neuroinflammation during normal aging in WT mice and in response to AD-associated pathology in APP/PS1dE9 Tg mice, an AD mouse model. Increased uptake and specific binding of 18F-GE180 in whole brain and hippocampus were confirmed by ex vivo PET and autoradiography. The binding specificity and stability of 18F-GE180 was further confirmed by a cold tracer competition study and a metabolite study, respectively. Therefore, 18F-GE180 PET imaging may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment and may also be useful for other neurodegenerative diseases.

An anti-pyroglutamate-3 Aβ vaccine reduces plaques and improves cognition in APPswe/PS1ΔE9 mice

Pyroglutamate-3 amyloid-beta (pGlu-3 Aβ) is an N-terminally truncated Aβ isoform likely playing a decisive role in Alzheimers disease pathogenesis. Here, we describe a prophylactic passive immunization study in APPswe/PS1ΔE9 mice using a novel pGlu-3 Aβ immunoglobulin G1 (IgG1) monoclonal antibody, 07/1 (150 and 500 μg, intraperitoneal, weekly) and compare its efficacy with a general Aβ IgG1 monoclonal antibody, 3A1 (200 μg, intraperitoneal, weekly) as a positive control. After 28 weeks of treatment, plaque burden was reduced and cognitive performance of 07/1-immunized Tg mice, especially at the higher dose, was normalized to wild-type levels in 2 hippocampal-dependent tests and partially spared compared with phosphate-buffered saline-treated Tg mice. Mice that received 3A1 had reduced plaque burden but showed no cognitive benefit. In contrast with 3A1, treatment with 07/1 did not increase the concentration of Aβ in plasma, suggesting different modes of Aβ plaque clearance. In conclusion, early selective targeting of pGlu-3 Aβ by immunotherapy may be effective in lowering cerebral Aβ plaque burden and preventing cognitive decline in the clinical setting. Targeting this pathologically modified form of Aβ thereby is unlikely to interfere with potential physiologic function(s) of Aβ that have been proposed.

Age-related epigenetic changes in hippocampal subregions of four animal models of Alzheimer's disease

Abstract Both aging and Alzheimers disease (AD) are associated with widespread epigenetic changes, with most evidence suggesting global hypomethylation in AD. It is, however, unclear how these age‐related epigenetic changes are linked to molecular aberrations as expressed in animal models of AD. Here, we investigated age‐related changes of epigenetic markers of DNA methylation and hydroxymethylation in a range of animal models of AD, and their correlations with amyloid plaque load. Three transgenic mouse models, including the J20, APP/PS1dE9 and 3xTg‐AD models, as well as Caribbean vervets (a non‐transgenic non‐human primate model of AD) were investigated. In the J20 mouse model, an age‐related decrease in DNA methylation was found in the dentate gyrus (DG) and a decrease in the ratio between DNA methylation and hydroxymethylation was found in the DG and cornu ammonis (CA) 3. In the 3xTg‐AD mice, an age‐related increase in DNA methylation was found in the DG and CA1‐2. No significant age‐related alterations were found in the APP/PS1dE9 mice and non‐human primate model. In the J20 model, hippocampal plaque load showed a significant negative correlation with DNA methylation in the DG, and with the ratio a negative correlation in the DG and CA3. For the APP/PS1dE9 model a negative correlation between the ratio and plaque load was observed in the CA3, as well as a negative correlation between DNA methyltransferase 3A (DNMT3A) levels and plaque load in the DG and CA3. Thus, only the J20 model showed an age‐related reduction in global DNA methylation, while DNA hypermethylation was observed in the 3xTg‐AD model. Given these differences between animal models, future studies are needed to further elucidate the contribution of different AD‐related genetic variation to age‐related epigenetic changes. HighlightsThe J20 mouse model shows an age‐related hippocampal decrease in DNA methylation.3xTg‐AD mice exhibit a hippocampal increase in DNA methylation with age.No epigenetic changes were detected in aging APP/PS1dE9 mice and Caribbean vervets.Differences between models suggest interaction genotype and epigenetic state.

Aging, biomarkers and behavior in Caribbean vervets

Background: Roughly 30,000 Caribbean vervets (African Green monkeys) live on the island of St. Kitts, Eastern Caribbean. The Behavioral Science Foundation has a colony of >1,000 vervets. Previously, we described the presence of amyloid-ß (Aß) plaques, vascular amyloid, neuritic dystrophy, and plaque-associated gliosis in a small number of aged vervets (Lemere et al., AJP, 2004). In this study, we extend our pathological analyses of brains and present correlations between aging, biomarkers, and behavior. Methods: Extensive immunohistochemical analysis was performed on archived, fixed sections from 28 vervets aged 12-32 yr to detect Aß deposition, gliosis, and p-tau. In addition, we have compared age, CSF Aß42, behavioral test scores (Object Retrieval Test for episodic memory, ORT), and biomarkers on human proteomic arrays (Rules Based Medicine) for each of 20 young (10 F, 10 M; ages 5-10 yr), 20 middle-aged (10 F, 10 M; ages >10-15 yr), and 20 old (10 F, 10 M; ages >15-26 yr) live vervets. Results: Aß deposition into plaques and/or blood vessels was observed in at least 1 brain region in all animals>17 yr and increased with age (P< 0.0008). Activated microglia and reactive astrocytes were associated with compacted plaques. Phosphotau immunoreactivity was localized to dystrophic plaque-associated neurites, neuropil threads, astrocytes, and very rare intracellular labeling. Cognitive decline was significantly associated with aging (young vs old, p < 0.02) however, some old vervets remained cognitively intact. CSF Aß42 increased with aging but in the old animals, there was a significant decrease in ?half of the animals that correlated with cognitive decline (p < 0.02) similar to changes seen in humans. Eighty-three of the 114 human markers used in the RBM Proteomics Array cross-reacted with vervet proteins. Early data analysis shows that increases in inflammatory proteins such as complement C3, IL-1ra, and CD40 ligand, as well as cortisol, correlated with worsening of episodic memory. However, of the four, only cortisol also showed significant positive correlations with age and CSF Aß42 levels. More extensive data analysis is underway. Conclusions: Vervets provide a valuable natural model for aging and at least some aspects of AD.