Jeffrey L. Frost

Galactic Cosmic Radiation Leads to Cognitive Impairment and Increased Aβ Plaque Accumulation in a Mouse Model of Alzheimer’s Disease

Galactic Cosmic Radiation consisting of high-energy, high-charged (HZE) particles poses a significant threat to future astronauts in deep space. Aside from cancer, concerns have been raised about late degenerative risks, including effects on the brain. In this study we examined the effects of 56Fe particle irradiation in an APP/PS1 mouse model of Alzheimer’s disease (AD). We demonstrated 6 months after exposure to 10 and 100 cGy 56Fe radiation at 1 GeV/µ, that APP/PS1 mice show decreased cognitive abilities measured by contextual fear conditioning and novel object recognition tests. Furthermore, in male mice we saw acceleration of Aβ plaque pathology using Congo red and 6E10 staining, which was further confirmed by ELISA measures of Aβ isoforms. Increases were not due to higher levels of amyloid precursor protein (APP) or increased cleavage as measured by levels of the β C-terminal fragment of APP. Additionally, we saw no change in microglial activation levels judging by CD68 and Iba-1 immunoreactivities in and around Aβ plaques or insulin degrading enzyme, which has been shown to degrade Aβ. However, immunohistochemical analysis of ICAM-1 showed evidence of endothelial activation after 100 cGy irradiation in male mice, suggesting possible alterations in Aβ trafficking through the blood brain barrier as a possible cause of plaque increase. Overall, our results show for the first time that HZE particle radiation can increase Aβ plaque pathology in an APP/PS1 mouse model of AD.

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.

Complement Component C3 and Complement Receptor Type 3 Contribute to the Phagocytosis and Clearance of Fibrillar Aβ by Microglia

Complement components and their receptors are found within and around amyloid β (Aβ) cerebral plaques in Alzheimers disease (AD). Microglia defend against pathogens through phagocytosis via complement component C3 and/or engagement of C3 cleavage product iC3b with complement receptor type 3 (CR3, Mac‐1). Here, we provide direct evidence that C3 and Mac‐1 mediate, in part, phagocytosis and clearance of fibrillar amyloid‐β (fAβ) by murine microglia in vitro and in vivo. Microglia took up not only synthetic fAβ42 but also amyloid cores from patients with AD, transporting them to lysosomes in vitro. Fibrillar Aβ42 uptake was significantly attenuated by the deficiency or knockdown of C3 or Mac‐1 and scavenger receptor class A ligands. In addition, C3 or Mac‐1 knockdown combined with a scavenger receptor ligand, fucoidan, further attenuated fibrillar Aβ42 uptake by N9 microglia. Fluorescent fibrillar Aβ42 microinjected cortically was significantly higher in C3 and Mac‐1 knockout mice compared with wild‐type mice 5 days after surgery, indicating reduced clearance in vivo. Together, these results demonstrate that C3 and Mac‐1 are involved in phagocytosis and clearance of fAβ by microglia, providing support for a potential beneficial role for microglia and the complement system in AD pathogenesis.

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.

Passive Immunization against Pyroglutamate-3 Amyloid-β Reduces Plaque Burden in Alzheimer-Like Transgenic Mice: A Pilot Study

Background: N-terminally truncated and modified pyroglutamate-3 amyloid-β protein (pE3-Aβ) is present in most, if not all, cerebral plaque and vascular amyloid deposits in human Alzheimer’s disease (AD). pE3-Aβ deposition is also found in AD-like transgenic (tg) mouse brain, albeit in lesser quantities than general Aβ. pE3-Aβ resists degradation, is neurotoxic, and may act as a seed for Aβ aggregation. Objective: We sought to determine if pE3-Aβ removal by passive immunization with a highly specific monoclonal antibody (mAb) impacts pathogenesis in a mouse model of Alzheimer’s amyloidosis. Methods: APPswe/PS1ΔE9 tg mice were given weekly intraperitoneal injections of a new anti-pE3-Aβ mAb (mAb07/1) or PBS from 5.8 to 13.8 months of age (prevention) or from 23 to 24.7 months of age (therapeutic). Multiple forms of cerebral Aβ were quantified pathologically and biochemically. Gliosis and microhemorrhage were examined. Results: Chronic passive immunization with an anti-pE3-Aβ mAb significantly reduced total plaque deposition and appeared to lower gliosis in the hippocampus and cerebellum in both the prevention and therapeutic studies. Insoluble Aβ levels in hemibrain homogenates were not significantly different between immunized and control mice. Microhemorrhage was not observed with anti-pE3-Aβ immunotherapy. Conclusions: Selective removal of pE3-Aβ lowered general Aβ plaque deposition suggesting a pro-aggregation or seeding role for pE3-Aβ.

Dietary Supplementation with S-Adenosyl Methionine Delayed Amyloid-β and Tau Pathology in 3xTg-AD Mice

S-adenosyl methionine (SAM) contributes to multiple pathways in neuronal homeostasis, several of which are compromised in age-related neurodegeneration and Alzheimers disease. Dietary supplementation of transgenic mice with SAM maintained acetylcholine levels, cognitive performance, oxidative buffering capacity, and phosphatase activity, and reduced aggression, calcium influx, endogenous PS-1 expression, γ-secretase activity, and levels of amyloid-β (Aβ) and phospho-tau. Herein, we examined whether or not SAM could delay neuropathology in 3xTg-AD mice, which harbor mutant genes for human AβPP, PS-1 and tau. Mice received a standard AIN-76 diet with or without SAM (100 mg/kg diet) for 1 month commencing at 10 months of age or for 3 months commencing at 12.5 months of age; mice were sacrificed and examined for Aβ and tau neuropathology at 11 and 15.5 months of age, respectively. SAM supplementation reduced hippocampal intracellular AβPP/Aβ and phospho-tau immunoreactivity to a similar extent at both sampling intervals. Supplementation reduced the number of extracellular Aβ deposits by 80% (p < 0.01) at 11 months of age after 1 month of treatment but only by 24% (p < 0.34) at 15.5 months of age after 3 months of treatment. As anticipated, neurofibrillary tangles were not observed in mice at these young ages; however, supplementation reduced levels of phospho-tau and caspase-cleaved tau within Sarkosyl-insoluble preparations in mice at 15.5 months of age. These limited analyses indicate that SAM can modulate the time course of AD neuropathology, and support further long-term analyses.

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.

MER5101, a Novel Aβ1-15:DT Conjugate Vaccine, Generates a Robust Anti-Aβ Antibody Response and Attenuates Aβ Pathology and Cognitive Deficits in APPswe/PS1ΔE9 Transgenic Mice

Active amyloid-β (Aβ) immunotherapy is under investigation to prevent or treat early Alzheimers disease (AD). In 2002, a Phase II clinical trial (AN1792) was halted due to meningoencephalitis in ∼6% of the AD patients, possibly caused by a T-cell-mediated immunological response. Thus, generating a vaccine that safely generates high anti-Aβ antibody levels in the elderly is required. In this study, MER5101, a novel conjugate of Aβ1-15 peptide (a B-cell epitope fragment) conjugated to an immunogenic carrier protein, diphtheria toxoid (DT), and formulated in a nanoparticular emulsion-based adjuvant, was administered to 10-month-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (Wt) mice. High anti-Aβ antibody levels were observed in both vaccinated APPswe/PS1ΔE9 Tg and Wt mice. Antibody isotypes were mainly IgG1 and IgG2b, suggesting a Th2-biased response. Restimulation of splenocytes with the Aβ1-15:DT conjugate resulted in a strong proliferative response, whereas proliferation was absent after restimulation with Aβ1-15 or Aβ1-40/42 peptides, indicating a cellular immune response against DT while avoiding an Aβ-specific T-cell response. Moreover, significant reductions in cerebral Aβ plaque burden, accompanied by attenuated microglial activation and increased synaptic density, were observed in MER5101-vaccinated APPswe/PS1ΔE9 Tg mice compared with Tg adjuvant controls. Last, MER5101-immunized APPswe/PS1ΔE9 Tg mice showed improvement of cognitive deficits in both contextual fear conditioning and the Morris water maze. Our novel, highly immunogenic Aβ conjugate vaccine, MER5101, shows promise for improving Aβ vaccine safety and efficacy and therefore, may be useful for preventing and/or treating early AD.

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.

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.