Mouse Model Of Aβ Amyloidosis Research Articles

Decreased plasma nicotinamide and altered NAD+ metabolism in glial cells surrounding Aβ plaques in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and a leading cause of senile dementia. Amyloid-β (Aβ) accumulation triggers chronic neuroinflammation, initiating AD pathogenesis. Recent clinical trials for anti-Aβ immunotherapy underscore that blood-based biomarkers have significant advantages and applicability over conventional diagnostics and are an unmet clinical need. To further advance ongoing clinical trials and identify novel therapeutic targets for AD, developing additional plasma biomarkers closely associated with pathogenic mechanisms downstream of Aβ accumulation is critically important. To identify plasma metabolites reflective of neuroinflammation caused by Aβ pathology, we performed untargeted metabolomic analyses of the plasma by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and analyzed the potential roles of the identified metabolic changes in the brain neuroinflammatory response using the female App knock-in (AppNLGF) mouse model of Aβ amyloidosis. The CE-TOFMS analysis of plasma samples from female wild-type (WT) and AppNLGF mice revealed that plasma levels of nicotinamide, a nicotinamide adenine dinucleotide (NAD+) precursor, were decreased in AppNLGF mice, and altered metabolite profiles were enriched for nicotinate/nicotinamide metabolism. In AppNLGF mouse brains, NAD+ levels were unaltered, but mRNA levels of NAD+-synthesizing nicotinate phosphoribosyltransferase (Naprt) and NAD+-degrading Cd38 genes were increased. These enzymes were induced in reactive astrocytes and microglia surrounding Aβ plaques in the cortex and hippocampus of female AppNLGF mouse brains, suggesting neuroinflammation increases NAD+ metabolism. This study suggests plasma nicotinamide could be indicative of the neuroinflammatory response and that nicotinate and nicotinamide metabolism are potential therapeutic targets for AD, by targeting both neuroinflammation and neuroprotection.

Effects of SPI1-mediated transcriptome remodeling on Alzheimer’s disease-related phenotypes in mouse models of Aβ amyloidosis

SPI1 was recently reported as a genetic risk factor for Alzheimer’s disease (AD) in large-scale genome-wide association studies. However, it is unknown whether SPI1 should be downregulated or increased to have therapeutic benefits. To investigate the effect of modulating SPI1 levels on AD pathogenesis, we performed extensive biochemical, histological, and transcriptomic analyses using both Spi1-knockdown and Spi1-overexpression mouse models. Here, we show that the knockdown of Spi1 expression significantly exacerbates insoluble amyloid-β (Aβ) levels, amyloid plaque deposition, and gliosis. Conversely, overexpression of Spi1 significantly ameliorates these phenotypes and dystrophic neurites. Further mechanistic studies using targeted and single-cell transcriptomics approaches demonstrate that altered Spi1 expression modulates several pathways, such as immune response pathways and complement system. Our data suggest that transcriptional reprogramming by targeting transcription factors, like Spi1, might hold promise as a therapeutic strategy. This approach could potentially expand the current landscape of druggable targets for AD.

Translational profiling identifies sex-specific metabolic and epigenetic reprogramming of cortical microglia/macrophages in APPPS1-21 mice with an antibiotic-perturbed-microbiome

BackgroundMicroglia, the brain-resident macrophages perform immune surveillance and engage with pathological processes resulting in phenotype changes necessary for maintaining homeostasis. In preceding studies, we showed that antibiotic-induced perturbations of the gut microbiome of APPPS1-21 mice resulted in significant attenuation in Aβ amyloidosis and altered microglial phenotypes that are specific to male mice. The molecular events underlying microglial phenotypic transitions remain unclear. Here, by generating ‘APPPS1-21-CD11br’ reporter mice, we investigated the translational state of microglial/macrophage ribosomes during their phenotypic transition and in a sex-specific manner.MethodsSix groups of mice that included WT-CD11br, antibiotic (ABX) or vehicle-treated APPPS1-21-CD11br males and females were sacrificed at 7-weeks of age (n = 15/group) and used for immunoprecipitation of microglial/macrophage polysomes from cortical homogenates using anti-FLAG antibody. Liquid chromatography coupled to tandem mass spectrometry and label-free quantification was used to identify newly synthesized peptides isolated from polysomes.ResultsWe show that ABX-treatment leads to decreased Aβ levels in male APPPS1-21-CD11br mice with no significant changes in females. We identified microglial/macrophage polypeptides involved in mitochondrial dysfunction and altered calcium signaling that are associated with Aβ-induced oxidative stress. Notably, female mice also showed downregulation of newly-synthesized ribosomal proteins. Furthermore, male mice showed an increase in newly-synthesized polypeptides involved in FcγR-mediated phagocytosis, while females showed an increase in newly-synthesized polypeptides responsible for actin organization associated with microglial activation. Next, we show that ABX-treatment resulted in substantial remodeling of the epigenetic landscape, leading to a metabolic shift that accommodates the increased bioenergetic and biosynthetic demands associated with microglial polarization in a sex-specific manner. While microglia in ABX-treated male mice exhibited a metabolic shift towards a neuroprotective phenotype that promotes Aβ clearance, microglia in ABX-treated female mice exhibited loss of energy homeostasis due to persistent mitochondrial dysfunction and impaired lysosomal clearance that was associated with inflammatory phenotypes.ConclusionsOur studies provide the first snapshot of the translational state of microglial/macrophage cells in a mouse model of Aβ amyloidosis that was subject to ABX treatment. ABX-mediated changes resulted in metabolic reprogramming of microglial phenotypes to modulate immune responses and amyloid clearance in a sex-specific manner. This microglial plasticity to support neuro-energetic homeostasis for its function based on sex paves the path for therapeutic modulation of immunometabolism for neurodegeneration.

Alzheimer’s disease biomarker‐related phospho‐tau 181 signals localize to demyelinated axons of parvalbumin‐positive GABAergic interneurons in App knock‐in mouse

AbstractBackgroundTau proteins phosphorylated at Thr181 (p‐tau 181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer’s disease (AD). An increase in p‐tau 181 level well correlates with Aβ pathology and precedes neurofibrillary tangle formation in the early stage of AD, though the relationship between p‐tau 181 and Aβ‐mediated pathology is less well understood. We have recently reported that p‐tau 181 signals are detected in the axonal structures and represent axonal abnormality around Aβ plaques in the cortex of App knock‐in mouse model of Aβ amyloidosis (AppNLGF mice). However, from which neuronal subtype(s) these p‐tau 181‐positive axons are originated remains elusive. In this study, we aimed to determine which neuronal subtype(s) express p‐tau 181 in the axons.MethodTo investigate the localization of p‐tau 181 signals in the brains of aged (24‐month‐old) AppNLGF and wild‐type mice, we prepared frozen brain sections and co‐immunostained with antibodies against p‐tau 181 and myeline basic protein (MBP), the glutamatergic neuronal vesicle marker vesicular glutamate transporter 1 (VGLUT1), the GABAergic neuronal vesicle marker vesicular GABA transporter (VGAT), the inhibitory neuron marker parvalbumin, the cholinergic neuronal vesicle marker vesicular acetylcholine transporter (VAChT), or the noradrenergic neuronal vesicle marker norepinephrine transporter (NET).ResultHistochemical analyses revealed that p‐tau 181 signals were not overlapped with axons of unmyelinated cholinergic nor noradrenergic neurons, but well colocalized with myelinated axons of parvalbumin‐positive GABAergic interneurons, but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, while that of glutamatergic, GABAergic or p‐tau 181‐positive axons was less affected. Instead, myelin sheaths surrounding p‐tau 181‐positive axons were significantly reduced in AppNLGF mice.ConclusionThis study suggests that p‐tau 181 signals may represent axonal abnormality of parvalbumin‐positive GABAergic interneurons with disrupted myelin sheaths in brains of mouse model of Aβ pathology.

Deletion of microRNA‐21 exacerbates multiple phenotypes associated with Alzheimer’s disease through the RECK and ADAM10 pathway in a mouse model of Aβ amyloidosis

AbstractBackgroundmicroRNAs have emerged as a new class of therapeutic targets that regulate the expression of target genes by binding their 3’‐untranslated regions. Growing evidence suggests that dysregulation of microRNAs may contribute to the onset and/or progression of various diseases. However, the functional and therapeutic implications of microRNA dysregulation in Alzheimer’s disease (AD) remain largely unclear. To gain insight into the roles of microRNAs in AD pathogenesis, we recently performed unbiased microRNA profiling with entorhinal cortices of AD and age‐matched non‐demented subjects. We found that miRNA‐21 (miR‐21) expression was markedly increased in AD patients. In addition, miR‐21 levels were increased in oligomeric amyloid‐beta (Aβ)‐treated neurons, suggesting that miR‐21 induction is directly associated with Aβ pathology. Therefore, we investigated the pathogenic/therapeutic potential of miR‐21 in AD.MethodTo investigate the role of miR‐21 in amyloid pathology in vivo, we crossed APP/PS1 mice with mir‐21−/− mice to generate APP/PS1;mir‐21+/+, APP/PS1;mir‐21+/− and, APP/PS1;mir‐21−/− mice. We evaluated the changes in the pathological features of AD, such as Aβ levels, amyloid plaques, and gliosis in these mice. Moreover, to investigate the effects of miR‐21 deletion on spatial learning and memory functions, we performed the Morris Water Maze (MWM) and Contextual fear conditioning (CFC) tests.ResultDeletion of miR‐21 increased the levels of insoluble and soluble Aβ40 and Aβ42 levels, as well as increased Aβ‐positive and X‐34‐positive plaque deposition in the cortex and hippocampus. CD45‐positive, IBA1‐positive microgliosis, and GFAP‐positive astrogliosis were also increased in the brain of APP/PS1;mir‐21+/− and APP/PS1;mir‐21−/− mice compared to APP/PS1;mir‐21+/+ mice. Mechanistic studies identified RECK as a potential target gene of miR‐21. miR‐21 deficiency upregulated the RECK protein level, leading to the inhibition of ADAM10 activity. This finding was also corroborated by the decrease in the soluble APPα level. Furthermore, we demonstrated that miR‐21 deficiency decreased spatial learning and contextual memory in MWM and CFC tests, respectively in APP/PS1 mice.ConclusionTaken together, our results demonstrated that miR‐21 may regulate multiple AD‐associated pathologies. Therefore, modulation of miR‐21 may represent a novel potential therapeutic strategy for AD.

Nanoelectrochemistry reveals how soluble Aβ42 oligomers alter vesicular storage and release of glutamate

Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by β-amyloid (Aβ) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aβ42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aβ42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aβ amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aβ42-induced neuronal hyperactivity and neuronal degeneration.

The pharmaceutical potential of AR‐65, a novel Alzheimer’s disease drug targeting the immunoproteasome

AbstractBackgroundPreviously, we reported that inhibition of the immunoproteasome (iP), which is upregulated in reactive glial cells near amyloid‐β (Aβ) deposits in the brains of patients with Alzheimer’s disease (AD), leads to improved cognitive function in an AD mouse model of Aβ amyloidosis (Tg2576) via suppression of microglia‐mediated inflammation, independent of Aβ accumulation.MethodHere, we investigated the pharmaceutical properties of our lead iP inhibitor (AR‐65), a macrocyclic peptide epoxyketone, selected based on in vitro and cellulo iP inhibitory activities and cell‐based anti‐inflammatory efficacy. Specifically, the metabolic stability of AR‐65 was assessed using pooled human liver microsomes. In addition, the flux rate of AR‐65 was measured to evaluate the permeability across Caco‐2 monolayers over‐expressing ABCB1 and ABCG2, two major efflux drug transporters present in the human brain.ResultThe macrocyclic AR‐65 displayed improved metabolic stability (6‐fold increased half‐life), permeability, and solubility (in an injectable pharmaceutical buffer system with no cyclodextrin) compared to the FDA‐approved linear peptide epoxyketone proteasome inhibitor, carfilzomib (Kyprolis®). Finally, pilot PK studies performed using ICR mice confirmed that AR‐65 is metabolically more stable than carfilzomib and can cross the blood‐brain barrier to inhibit the brain iP.ConclusionTaken together, the results suggest that AR‐65 may hold great potential to be further investigated in clinical settings.

Reduced density of cholinergic fibers in App knock‐in mouse models of Aβ amyloidosis

AbstractBackgroundCholinergic system is critical for learning, memory, attention, and other cognitive functions. Nucleus basalis of Meynert (NBM) is a nucleus containing the cell bodies of cholinergic neurons in the brain and is severely affected by Alzheimer’s disease (AD). Significant loss of forebrain cholinergic projections is evident at prodromal stage of AD, and, along with locus coeruleus (LC), NBM neurons are among the first to display neurofibrillary tangle, suggesting that abnormality in cholinergic system is involved in the early stage of AD pathogenesis. Acetylcholinesterase inhibitors have been used for symptomatic treatment to improve cognitive function, however, mechanisms underlying neurodegeneration in cholinergic system remain elusive. We have previously reported that AppNL‐G‐F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited cognitive deficits, severe neuroinflammation and significant loss of LC noradrenergic fibers accompanied by massive amyloid‐β (Aβ) pathology. In this study, we asked whether and how cholinergic system is altered in AppNL‐G‐F mice.MethodTo ask whether AppNL‐G‐F mice exhibit cholinergic degeneration, we compared the density of vesicular acetylcholine transporter (VAChT)‐positive fibers in the cortex and hippocampus as well as the number of choline acetyltransferase (ChAT)‐positive neurons in the NBM between AppNL‐G‐F and wild‐type (WT) C57BL/6J mice at 24 months of age. We also examined whether tau pathology occurs in the cholinergic system in these mice.ResultHistochemical analyses revealed that 24‐month‐old AppNL‐G‐F mice exhibited significant decreases in the density of VAChT‐positive fibers in the cortex and the hippocampal CA1, the regions associated with learning and memory, compared to WT mice.ConclusionThis study demonstrates that Aβ pathology is sufficient to reduce the density of cholinergic fibers. Quantitative analyses of cell loss and tau accumulation in ChAT‐positive neurons in the NBM are currently underway, and we will present these results at the meeting.

Localization of AD biomarker phospho-tau proteins in the brains of an App knock-in mouse model of Aβ amyloidosis.

Amyloid-β (Aβ) plaques, neurofibrillary tangles composed of hyper-phosphorylated tau proteins and neurodegeneration are neuropathological hallmarks of Alzheimer's disease (AD). Recently, phospho-tau proteins in blood as well as cerebrospinal fluid have been reported as promising biomarkers in the preclinical stage of AD. However, physiological and pathological roles of these phosphorylated tau proteins in AD pathogenesis remain elusive. In this study, we investigated the localization of AD biomarker phospho-tau proteins in the brains of App knock-in (App NLGF ) mouse, which is thought to mimic important aspects in the early phase of AD. To investigate the localization of AD biomarker phospho-tau proteins in the brains of aged (24- month-old) App NLGF and wild-type mice, we prepared frozen brain sections and co-immunostained with phospho-tau specific antibodies and reactive gliosis or synaptic marker antibodies. We found that one AD biomarker phospho-tau signals were detected in the axonal structures in the brains of wild-type mice, and these structures were disrupted in the brains of App NLGF mice. In contrast, the other AD biomarker phospho-tau epitopes were only detected in the brains of App NLGF mice as punctate signals around Aβ plaques. These phospho-tau signals were co-localized with post-synaptic marker PSD95, but not with reactive gliosis or pre-synaptic markers. Our results suggest that AD biomarker phospho-tau epitopes may represent axonal degeneration or Aβ plaque-related post-synaptic pathology in the early phase of AD brains.

Deletion of Abi3 gene locus exacerbates neuropathological features of Alzheimer's disease in a mouse model of Aβ amyloidosis.

Recently, large-scale human genetics studies identified a rare coding variant in the ABI3 gene that is associated with an increased risk of Alzheimer’s disease (AD). However, pathways by which ABI3 contributes to the pathogenesis of AD are unknown. To address this question, we determined whether loss of ABI3 function affects pathological features of AD in the 5XFAD mouse model. We demonstrate that the deletion of Abi3 locus significantly increases amyloid β (Aβ) accumulation and decreases microglia clustering around the plaques. Furthermore, long-term potentiation is impaired in 5XFAD;Abi3 knockout (“Abi3−/−”) mice. Moreover, we identified marked changes in the proportion of microglia subpopulations in Abi3−/− mice using a single-cell RNA sequencing approach. Mechanistic studies demonstrate that Abi3 knockdown in microglia impairs migration and phagocytosis. Together, our study provides the first in vivo functional evidence that loss of ABI3 function may increase the risk of developing AD by affecting Aβ accumulation and neuroinflammation.

Comparison of memory, affective behavior, and neuropathology in APPNLGF knock-in mice to 5xFAD and APP/PS1 mice

Transgenic mouse models of Aβ amyloidosis generated by knock-in of a humanized Aβ sequence can offer some advantages over the transgenic models that overexpress amyloid precursor protein (APP). However, systematic comparison of memory, behavioral, and neuropathological phenotypes between these models has not been well documented. In this study, we compared memory and affective behavior in APPNLGF mice, an APP knock-in model, to two widely used mouse models of Alzheimer’s disease, 5xFAD and APP/PS1 mice, at 10 months of age. We found that, despite similar deficits in working memory, object recognition, and social recognition memory, APPNLGF and 5xFAD mice but not APP/PS1 mice show compelling anxiety- and depressive-like behavior, and exhibited a marked impairment of social interaction. We quantified corticolimbic Aβ plaques, which were lowest in APPNLGF, intermediate in APP/PS1, and highest in 5xFAD mice. Interestingly, analysis of plaque size revealed that plaques were largest in APP/PS1 mice, intermediate in 5xFAD mice, and smallest in APPNLGF mice. Finally, we observed a significantly higher percentage of the area occupied by plaques in both 5xFAD and APP/PS1 relative to APPNLGF mice. Overall, our findings suggest that the severity of Aβ neuropathology is not directly correlated with memory and affective behavior impairments between these three transgenic mouse models. Additionally, APPNLGF may represent a valid mouse model for studying AD comorbid with anxiety and depression.

Reduced density of noradrenergic fibers without prominent neuron loss or tau pathology in the locus coeruleus in APP knock‐in mouse models of Aβ amyloidosis

AbstractBackgroundLocus coeruleus (LC) is a nucleus containing the cell bodies of noradrenergic neurons in the brain, and is one of the earliest regions affected by Alzheimer’s disease (AD). Norepinephrine (NE) is critical for cognitive functions as well as anti‐inflammatory and neuroprotective properties, both of which are implicated in AD. LC neurons are among the first to display tau pathology and that significant loss of forebrain noradrenergic projections is evident at prodromal stage of AD, suggesting that abnormality in LC‐NE system initiates the disease pathogenesis. However, mechanisms underlying neurodegeneration in LC‐NE system remain elusive. We have previously reported that AppNL‐G‐F/NL‐G‐F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited cognitive deficits and severe neuroinflammation accompanied by massive amyloid‐β (Aβ) pathology. In this study, we asked whether and how LC‐NE system is altered in AppNL‐G‐F/NL‐G‐F mice.MethodTo ask whether AppNL‐G‐F/NL‐G‐F mice exhibit noradrenergic degeneration, we compared the density of norepinephrine transporter (NET)‐positive fibers in the cortex and hippocampus as well as the number of dopamine β‐hydroxylase (DBH)‐positive neurons in the LC between AppNL‐G‐F/NL‐G‐F and wild‐type (WT) C57BL/6J mice at 24 months of age. We also examined whether tau pathology occurs in the LC‐NE system in these mice.ResultHistochemical analyses revealed that 24‐month‐old AppNL‐G‐F/NL‐G‐F mice exhibited significant decreases in the density of NET‐positive fibers in the prefrontal and entorhinal cortices and the hippocampal CA1, the regions associated with learning and memory, compared to WT mice. In contrast, AppNL‐G‐F/NL‐G‐F mice did not display loss of DBH‐positive neurons in the LC even at 24 months of age. Furthermore, AppNL‐G‐F/NL‐G‐F mice did not develop prominent tau pathology either in the nerve terminals or in the cell bodies of LC neurons.ConclusionThis study demonstrates that Aβ pathology is sufficient to reduce the density of noradrenergic fibers independent of neuron loss and tau pathology in the LC. These results also suggest that age‐dependent accumulation of Aβ in the cortex or tau in the LC may additively and independently damage LC neurons from the nerve terminals or from the cell bodies, respectively.

Development of a PET radioligand selective for cerebral amyloid angiopathy

IntroductionPositron emission tomography (PET) using radiolabeled amyloid-binding compounds has advanced the field of Alzheimer's disease (AD) by enabling detection and longitudinal tracking of fibrillar amyloid-β (Aβ) deposits in living people. However, this technique cannot distinguish between Aβ deposits in brain parenchyma (amyloid plaques) from those in blood vessels (cerebral amyloid angiopathy, CAA). Development of a PET radioligand capable of selectively detecting CAA would help clarify its contribution to global brain amyloidosis and clinical symptoms in AD and would help to characterize side-effects of anti-Aβ immunotherapies in AD patients, such as CAA. MethodsA candidate CAA-selective compound (1) from a panel of analogues of the amyloid-binding dye Congo red was synthesized. The binding affinity to Aβ fibrils and lipophilicity of compound 1 were determined and selectivity for CAA versus parenchymal plaque deposits was assessed ex-vivo and in-vivo in transgenic APP/PS1 mice and in postmortem human brain affected with AD pathology. ResultsCompound 1 displays characteristics of Aβ binding dyes, such as thioflavin-S, in that it labels both parenchymal Aβ plaques and CAA when applied to histological sections from both a transgenic APP/PS1 mouse model of Aβ amyloidosis and AD brain. Thus, compound 1 lacks molecular selectivity to distinguish Aβ deposits in CAA from those in plaques. However, when administered to living APP/PS1 mice intravenously, compound 1 preferentially labels CAA when assessed using in-vivo two-photon microscopy and ex-vivo histology and autoradiography. ConclusionWe hypothesize that selectivity of compound 1 for CAA is attributable to its limited penetration of the blood-brain barrier due to the highly polar nature of the carboxylate moiety, thereby limiting access to parenchymal plaques and promoting selective in-vivo labeling of Aβ deposits in the vascular wall (i.e., “delivery selectivity”).

Sex-specific effects of microbiome perturbations on cerebral Aβ amyloidosis and microglia phenotypes.

We demonstrated that an antibiotic cocktail (ABX)-perturbed gut microbiome is associated with reduced amyloid-β (Aβ) plaque pathology and astrogliosis in the male amyloid precursor protein (APP)SWE /presenilin 1 (PS1)ΔE9 transgenic model of Aβ amyloidosis. We now show that in an independent, aggressive APPSWE/PS1L166P (APPPS1-21) mouse model of Aβ amyloidosis, an ABX-perturbed gut microbiome is associated with a reduction in Aβ pathology and alterations in microglial morphology, thus establishing the generality of the phenomenon. Most importantly, these latter alterations occur only in brains of male mice, not in the brains of female mice. Furthermore, ABX treatment lead to alterations in levels of selected microglial expressed transcripts indicative of the "M0" homeostatic state in male but not in female mice. Finally, we found that transplants of fecal microbiota from age-matched APPPS1-21 male mice into ABX-treated APPPS1-21 male restores the gut microbiome and partially restores Aβ pathology and microglial morphology, thus demonstrating a causal role of the microbiome in the modulation of Aβ amyloidosis and microglial physiology in mouse models of Aβ amyloidosis.

Cognitive and emotional alterations in App knock-in mouse models of A\u03b2 amyloidosis

BackgroundAlzheimer’s disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Mouse models of Aβ amyloidosis generated by knock-in (KI) of a humanized Aβ sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aβ pathology observed in AD: the Swedish (NL) mutation, which elevates total Aβ production; the Beyreuther/Iberian (F) mutation, which increases the Aβ42/Aβ40 ratio; and the Arctic (G) mutation, which promotes Aβ aggregation. AppNL-G-F mice harbor all three mutations and develop progressive Aβ amyloidosis and neuroinflammatory response in broader brain areas, whereas AppNL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aβ pathology, we assessed emotional and cognitive domains of AppNL-G-F and AppNL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks.ResultsAssessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, AppNL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, AppNL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both AppNL/NL and AppNL-G-F/NL-G-F mice exhibited intact learning and memory up to 15–18 months of age, whereas AppNL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, AppNL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions.ConclusionAppNL/NL and AppNL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that AppNL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration.

Cannabinoid CB2 Receptors in a Mouse Model of Aβ Amyloidosis: Immunohistochemical Analysis and Suitability as a PET Biomarker of Neuroinflammation.

In Alzheimer’s disease (AD), one of the early responses to Aβ amyloidosis is recruitment of microglia to areas of new plaque. Microglial receptors such as cannabinoid receptor 2 (CB2) might be a suitable target for development of PET radiotracers that could serve as imaging biomarkers of Aβ-induced neuroinflammation. Mouse models of amyloidosis (J20APPswe/ind and APPswe/PS1ΔE9) were used to investigate the cellular distribution of CB2 receptors. Specificity of CB2 antibody (H60) was confirmed using J20APPswe/ind mice lacking CB2 receptors. APPswe/PS1ΔE9 mice were used in small animal PET with a CB2-targeting radiotracer, [11C]A836339. These studies revealed increased binding of [11C]A836339 in amyloid-bearing mice. Specificity of the PET signal was confirmed in a blockade study with a specific CB2 antagonist, AM630. Confocal microscopy revealed that CB2-receptor immunoreactivity was associated with astroglial (GFAP) and, predominantly, microglial (CD68) markers. CB2 receptors were observed, in particular, in microglial processes forming engulfment synapses with Aβ plaques. In contrast to glial cells, neuron (NeuN)-derived CB2 signal was equal between amyloid-bearing and control mice. The pattern of neuronal CB2 staining in amyloid-bearing mice was similar to that in human cases of AD. The data collected in this study indicate that Aβ amyloidosis without concomitant tau pathology is sufficient to activate CB2 receptors that are suitable as an imaging biomarker of neuroinflammation. The main source of enhanced CB2 PET binding in amyloid-bearing mice is increased CB2 immunoreactivity in activated microglia. The presence of CB2 immunoreactivity in neurons does not likely contribute to the enhanced CB2 PET signal in amyloid-bearing mice due to a lack of significant neuronal loss in this model. However, significant loss of neurons as seen at late stages of AD might decrease the CB2 PET signal due to loss of neuronally-derived CB2. Thus this study in mouse models of AD indicates that a CB2-specific radiotracer can be used as a biomarker of neuroinflammation in the early preclinical stages of AD, when no significant neuronal loss has yet developed.

Anti-ApoE antibody given after plaque onset decreases Aβ accumulation and improves brain function in a mouse model of Aβ amyloidosis.

Apolipoprotein E (apoE) is the strongest known genetic risk factor for late onset Alzheimer's disease (AD). It influences amyloid-β (Aβ) clearance and aggregation, which likely contributes in large part to its role in AD pathogenesis. We recently found that HJ6.3, a monoclonal antibody against apoE, significantly reduced Aβ plaque load when given to APPswe/PS1ΔE9 (APP/PS1) mice starting before the onset of plaque deposition. To determine whether the anti-apoE antibody HJ6.3 affects Aβ plaques, neuronal network function, and behavior in APP/PS1 mice after plaque onset, we administered HJ6.3 (10 mg/kg/week) or PBS intraperitoneally to 7-month-old APP/PS1 mice for 21 weeks. HJ6.3 mildly improved spatial learning performance in the water maze, restored resting-state functional connectivity, and modestly reduced brain Aβ plaque load. There was no effect of HJ6.3 on total plasma cholesterol or cerebral amyloid angiopathy. To investigate the underlying mechanisms of anti-apoE immunotherapy, HJ6.3 was applied to the brain cortical surface and amyloid deposition was followed over 2 weeks using in vivo imaging. Acute exposure to HJ6.3 affected the course of amyloid deposition in that it prevented the formation of new amyloid deposits, limited their growth, and was associated with occasional clearance of plaques, a process likely associated with direct binding to amyloid aggregates. Topical application of HJ6.3 for only 14 d also decreased the density of amyloid plaques assessed postmortem. Collectively, these studies suggest that anti-apoE antibodies have therapeutic potential when given before or after the onset of Aβ pathology.

Treatment with bexarotene, a compound that increases apolipoprotein-E, provides no cognitive benefit in mutant APP/PS1 mice

BackgroundThough the precise cause(s) of Alzheimer’s disease (AD) remain unknown, there is strong evidence that decreased clearance of β-amyloid (Aβ) from the brain can contribute to the disease. Therapeutic strategies to promote natural Aβ clearance mechanisms, such as the protein apolipoprotein-E (APOE), hold promise for the treatment of AD. The amount of APOE in the brain is regulated by nuclear receptors including retinoid X receptors (RXRs). Drugs that activate RXRs, including bexarotene, can increase APOE and ABCA1 production, and have been shown to decrease the Aβ burden and improve cognition in mouse models of Aβ amyloidosis. Although recent bexarotene studies failed to replicate the rapid clearance of Aβ from brains, behavioral and cognitive effects of this compound remain controversial.FindingsIn efforts to clarify these behavioral findings, mutant APP/PS1 mice were acutely dosed with bexarotene. While ABCA1 was upregulated in mutant APP/PS1 mice treated with bexarotene, this drug failed to attenuate Aβ plaques or cognitive deficits in these mice.ConclusionsWe recommend rigorous preclinical study to evaluate the mechanism and utility of such a compound for AD therapy.

Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aβ amyloidosis

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer's disease (AD). The influence of apoE on amyloid β (Aβ) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aβ and facilitates Aβ fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aβ antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60-80% and significantly reduced insoluble Aβ40 and Aβ42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aβ plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aβ binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases.

Aging diminishes neuronal transport in wild-type mice, but not in an accelerated mouse model of Aβ amyloidosis

Manganese-Enhanced MRI (MEMRI) allows for a non-invasive in vivo measurement of neuronal transport. Previous MEMRI studies on an animal model of Aß deposition (Tg2576 mice) have demonstrated that the expression of human mutant APP induces in vivo perturbations of neuronal transport. The aim of our study was to characterize neuronal transport in an accelerated mouse model of Aß amyloidosis, which expresses 5 mutations of APP and PS1 human genes (5XFAD). Thirteen Tg and 15 WT mice were imaged on a 7T magnet at 3 or 6 months of age. We used a tract-tracing MEMRI protocol with 9 imaging time points, 1 prior to and 8 following nasal injection of MnCl2. Two regions of interest (ROI) were defined on MR images, corresponding to the glomerular layer and the mitral cell layer of the olfactory bulb. In each ROI, the evolution of signal intensity profile was used to estimate the maximum (Smax), maximal slope (Vmax) and time to maximal slope (T2Vmax) of the relative manganese concentration. Unexpectedly, WT mice demonstrated an age-associated impairment of neuronal transport, assessed by a decrease in Smax (p < 0.01), decrease in Vmax (p < 0.01) and increase in T2Vmax (p < 0.005) between 3 and 6 months of age. Surprisingly, this age-related impairment was lacking in the Tg group, in which neuronal transport parameters remained stable between 3 and 6 months of age. At 6 months, T2Vmax was significantly lower in the mitral cell layer of Tg mice as compared to WT controls (p < 0.05), suggesting a faster neuronal transport in the Tg group.