Microglial cathepsin E plays a role in neuroinflammation and amyloid β production in Alzheimer’s disease

Regulation of neuroinflammation and β‐amyloid (Aβ) production are critical factors in the pathogenesis of Alzheimer''s disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock‐in mice, called APP^NL−G−F mice, significantly reduced Aβ accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF‐related apoptosis‐inducing ligand, which plays an important role in microglia‐mediated NF‐κB‐dependent neuroinflammation and neuronal Aβ production by beta‐site APP cleaving enzyme 1. Furthermore, cannula‐delivered CatE inhibitors improved memory function and reduced Aβ accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aβ pathology.     

TRPV1 sustains microglial metabolic reprogramming in Alzheimer's disease

As the brain‐resident innate immune cells, reactive microglia are a major pathological feature of Alzheimer''s disease (AD). However, the exact role of microglia is still unclear in AD pathogenesis. Here, using metabolic profiling, we show that microglia energy metabolism is significantly suppressed during chronic Aβ‐tolerant processes including oxidative phosphorylation and aerobic glycolysis via the mTOR‐AKT‐HIF‐1α pathway. Pharmacological activation of TRPV1 rescues Aβ‐tolerant microglial dysfunction, the AKT/mTOR pathway activity, and metabolic impairments and restores the immune responses including phagocytic activity and autophagy function. Amyloid pathology and memory impairment are accelerated in microglia‐specific TRPV1‐knockout APP/PS1 mice. Finally, we showed that metabolic boosting with TRPV1 agonist decreases amyloid pathology and reverses memory deficits in AD mice model. These results indicate that TRPV1 is an important target regulating metabolic reprogramming for microglial functions in AD treatment.     

Intranasal insulin alleviates cognitive deficits and amyloid pathology in young adult APPswe/PS1dE9 mice

Brain insulin signaling deficits contribute to multiple pathological features of Alzheimer's disease (AD). Although intranasal insulin has shown efficacy in patients with AD, the underlying mechanisms remain largely unillustrated. Here, we demonstrate that intranasal insulin improves cognitive deficits, ameliorates defective brain insulin signaling, and strongly reduces β‐amyloid (Aβ) production and plaque formation after 6 weeks of treatment in 4.5‐month‐old APPswe/PS1dE9 (APP/PS1) mice. Furthermore, c‐Jun N‐terminal kinase activation, which plays a pivotal role in insulin resistance and AD pathologies, is significantly inhibited. The alleviation of amyloid pathology by intranasal insulin results mainly from enhanced nonamyloidogenic processing and compromised amyloidogenic processing of amyloid precursor protein (APP), and from a reduction in apolipoprotein E protein which is involved in Aβ metabolism. In addition, intranasal insulin effectively promotes hippocampal neurogenesis in APP/PS1 mice. This study, exploring the mechanisms underlying the beneficial effects of intranasal insulin on Aβ pathologies in vivo for the first time, highlights important preclinical evidence that intranasal insulin is potentially an effective therapeutic method for the prevention and treatment of AD.     

Insulin resistance induces earlier initiation of cognitive dysfunction mediated by cholinergic deregulation in a mouse model of Alzheimer's disease

Although insulin resistance increases the risk of Alzheimer's disease (AD), the mechanisms remain unclear, partly because no animal model exhibits the insulin-resistant phenotype without persistent hyperglycemia. Here we established an AD model with whole-body insulin resistance without persistent hyperglycemia (APP/IR-dKI mice) by crossbreeding constitutive knock-in mice with P1195L-mutated insulin receptor (IR-KI mice) and those with mutated amyloid precursor protein (App^NL-G-F mice: APP-KI mice). APP/IR-dKI mice exhibited cognitive impairment at an earlier age than APP-KI mice. Since cholinergic dysfunction is a major characteristic of AD, pharmacological interventions on the cholinergic system were performed to investigate the mechanism. Antagonism to a nicotinic acetylcholine receptor α7 (nAChRα7) suppressed cognitive function and cortical blood flow (CBF) response to cholinergic-regulated peripheral stimulation in APP-KI mice but not APP/IR-dKI mice. Cortical expression of Chrna7, encoding nAChRα7, was downregulated in APP/IR-dKI mice compared with APP-KI. Amyloid β burden did not differ between APP-KI and APP/IR-dKI mice. Therefore, insulin resistance, not persistent hyperglycemia, induces the earlier onset of cognitive dysfunction and CBF deregulation mediated by nAChRα7 downregulation. Our mouse model will help clarify the association between type 2 diabetes mellitus and AD.     

Icariin Ameliorates Neuropathological Changes,TGF-β1 Accumulation and Behavioral Deficits in a Mouse Model of Cerebral Amyloidosis

Icariin, a major constituent of flavonoids from the Chinese medicinal herb Epimedium brevicornum, exhibits multiple biological properties, including anti-inflammatory, neuroregulatory and neuroprotective activities. Therefore, Icariin might be applied in treatment of neurodegenerative disorders, including Alzheimer''s disease (AD), which is neuropathologically characterized by β-amyloid aggregation, hyperphosphorylated tau and neuroinflammation. Potential therapeutic effects of Icariin were investigated in an animal model of cerebral amyloidosis for AD, transgenic APP/PS1 mouse. Icariin was suspended in carboxymethylcellulose and given orally to APP/PS1 mice. Therapeutic effects were monitored by behavioral tests, namely nesting assay, before and during the experimental treatment. Following an oral treatment of 10 days, Icariin significantly attenuated Aβ deposition, microglial activation and TGF-β1 immunoreactivity at amyloid plaques in cortex and hippocampus of transgenic mice 5 months of age, and restored impaired nesting ability. Our results suggest that Icariin might be considered a promising therapeutic option for human AD.     

Intracerebroventricular Streptozotocin Exacerbates Alzheimer-Like Changes of 3xTg-AD Mice

Alzheimer's disease (AD) involves several possible molecular mechanisms, including impaired brain insulin signaling and glucose metabolism. To investigate the role of metabolic insults in AD, we injected streptozotocin (STZ), a diabetogenic compound if used in the periphery, into the lateral ventricle of the 6-month-old 3xTg-AD mice and studied the cognitive function as well as AD-like brain abnormalities, such as tau phosphorylation and Aβ accumulation, 3–6 weeks later. We found that STZ exacerbated impairment of short-term and spatial reference memory in 3xTg-AD mice. We also observed an increase in tau hyperphosphorylation and neuroinflammation, a disturbance of brain insulin signaling, and a decrease in synaptic plasticity and amyloid β peptides in the brain after STZ treatment. The expression of 20 AD-related genes, including those involved in the processing of amyloid precursor protein, cytoskeleton, glucose metabolism, insulin signaling, synaptic function, protein kinases, and apoptosis, was altered, suggesting that STZ disturbs multiple metabolic and cell signaling pathways in the brain. These findings provide experimental evidence of the role of metabolic insult in AD.     

Oridonin ameliorates neuropathological changes and behavioural deficits in a mouse model of cerebral amyloidosis

Alzheimer's disease (AD) is the most common form of neurodegeneration and the major cause of dementia. This multifactorial disorder is clinically defined by progressive behavioural and cognitive deficits, and neuropathologically characterized by β‐amyloid aggregation, hyperphosphorylated tau and neuroinflammation. Oridonin, a diterpenoid isolated from Chinese herb Rabdosia rubescens, has multiple biological properties, especially anti‐inflammatory and neuroregulatory activities. Potential therapeutic effects of Oridonin were investigated in an animal model of cerebral amyloidosis for AD, transgenic APP/PS1 mice. Oridonin was suspended in carboxymethylcellulose or loaded with a nanostructured emulsion, and was orally administrated or injected. Before, during and following the experimental treatments, behavioural tests were performed with these transgenic mice and their naive littermates. Following relatively short‐term treatments of 10 days, brain tissue of mice were removed for immunohistochemical assays. The results indicate that both oral treatment and injection of Oridonin significantly attenuated β‐amyloid deposition, plaque‐associated APP expression and microglial activation in brain of transgenic mice. Furthermore, injection of Oridonin‐nanoemulsion ameliorated deficits in nesting, an important affiliative behaviour, and in social interaction. Additional in vitro studies indicated that Oridonin effectively attenuated inflammatory reaction of macrophage and microglial cell lines. Our results suggest that Oridonin might be considered a promising therapeutic option for human AD or other neurodegenerative diseases.     

Cooperative effects of SIRT1 and SIRT2 on APP acetylation

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) deposition and neurofibrillary tangles. Although the NAD⁺-dependent deacetylases SIRT1 and SIRT2 play pivotal roles in age-related diseases, their cooperative effects in AD have not yet been elucidated. Here, we report that the SIRT2:SIRT1 ratio is elevated in the brains of aging mice and in the AD mouse models. In HT22 mouse hippocampal neuronal cells, Aβ challenge correlates with decreased SIRT1 expression, while SIRT2 expression is increased. Overexpression of SIRT1 prevents Aβ-induced neurotoxicity. We find that SIRT1 impedes SIRT2-mediated APP deacetylation by inhibiting the binding of SIRT2 to APP. Deletion of SIRT1 reduces APP recycling back to the cell surface and promotes APP transiting toward the endosome, thus contributing to the amyloidogenic processing of APP. Our findings define a mechanism for neuroprotection by SIRT1 through suppression of SIRT2 deacetylation, and provide a promising avenue for therapeutic intervention of AD.     

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.     

Insulin resistance and amyloidogenesis as common molecular foundation for type 2 diabetes and Alzheimer's disease

Characterized as a peripheral metabolic disorder and a degenerative disease of the central nervous system respectively, it is now widely recognized that type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share several common abnormalities including impaired glucose metabolism, increased oxidative stress, insulin resistance and amyloidogenesis. Several recent studies suggest that this is not an epiphenomenon, but rather these two diseases disrupt common molecular pathways and each disease compounds the progression of the other. For instance, in AD the accumulation of the amyloid-beta peptide (Aβ), which characterizes the disease and is thought to participate in the neurodegenerative process, may also induce neuronal insulin resistance. Conversely, disrupting normal glucose metabolism in transgenic animal models of AD that over-express the human amyloid precursor protein (hAPP) promotes amyloid-peptide aggregation and accelerates the disease progression. Studying these processes at a cellular level suggests that insulin resistance and Aβ aggregation may not only be the consequence of excitotoxicity, aberrant Ca²⁺ signals, and proinflammatory cytokines such as TNF-α, but may also promote these pathological effectors. At the molecular level, insulin resistance and Aβ disrupt common signal transduction cascades including the insulin receptor family/PI3 kinase/Akt/GSK3 pathway. Thus both disease processes contribute to overlapping pathology, thereby compounding disease symptoms and progression.     

Pioglitazone Improves Reversal Learning and Exerts Mixed Cerebrovascular Effects in a Mouse Model of Alzheimer’s Disease with Combined Amyloid-β and Cerebrovascular Pathology

Animal models of Alzheimer’s disease (AD) are invaluable in dissecting the pathogenic mechanisms and assessing the efficacy of potential new therapies. Here, we used the peroxisome proliferator-activated receptor gamma agonist pioglitazone in an attempt to rescue the pathogenic phenotype in adult (12 months) and aged (>18 months) bitransgenic A/T mice that overexpress a mutated human amyloid precursor protein (APPSwe,Ind) and a constitutively active form of transforming growth factor-β1 (TGF-β1). A/T mice recapitulate the AD-related cognitive deficits, amyloid beta (Aβ) and cerebrovascular pathologies, as well as the altered metabolic and vascular coupling responses to increased neuronal activity. Pioglitazone normalized neurometabolic and neurovascular coupling responses to sensory stimulation, and reduced cortical astroglial and hippocampal microglial activation in both age groups. Spatial learning and memory deficits in the Morris water maze were not rescued by pioglitazone, but reversal learning was improved in the adult cohort notwithstanding a progressing Aβ pathology. While pioglitazone preserved the constitutive nitric oxide synthesis in the vessel wall, it unexpectedly failed to restore cerebrovascular reactivity in A/T mice and even exacerbated the dilatory deficits. These data demonstrate pioglitazone’s efficacy on selective AD hallmarks in a complex AD mouse model of comorbid amyloidosis and cerebrovascular pathology. They further suggest a potential benefit of pioglitazone in managing neuroinflammation, cerebral perfusion and glucose metabolism in AD patients devoid of cerebrovascular pathology.     

HDAC3 negatively regulates spatial memory in a mouse model of Alzheimer's disease

The accumulation and deposition of beta‐amyloid (Aβ) is a key neuropathological hallmark of Alzheimer's disease (AD). Histone deacetylases (HDACs) are promising therapeutic targets for the treatment of AD, while the specific HDAC isoforms associated with cognitive improvement are poorly understood. In this study, we investigate the role of HDAC3 in the pathogenesis of AD. Nuclear HDAC3 is significantly increased in the hippocampus of 6‐ and 9‐month‐old APPswe/PS1dE9 (APP/PS1) mice compared with that in age‐matched wild‐type C57BL/6 (B6) mice. Lentivirus ‐mediated inhibition or overexpression of HDAC3 was used in the hippocampus of APP/PS1 mice to investigate the role of HDAC3 in spatial memory, amyloid burden, dendritic spine density, glial activation and tau phosphorylation. Inhibition of HDAC3 in the hippocampus attenuates spatial memory deficits, as indicated in the Morris water maze test, and decreases amyloid plaque load and Aβ levels in the brains of APP/PS1 mice. Dendritic spine density is increased, while microglial activation is alleviated after HDAC3 inhibition in the hippocampus of 9‐month‐old APP/PS1 mice. Furthermore, HDAC3 overexpression in the hippocampus increases Aβ levels, activates microglia, and decreases dendritic spine density in 6‐month‐old APP/PS1 mice. In conclusion, our results indicate that HDAC3 negatively regulates spatial memory in APP/PS1 mice and HDAC3 inhibition might represent a potential therapy for the treatment of AD.     

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer''s disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer''s disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)¹, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology². The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.     

Glutathione Peroxidase 4 is associated with Neuromelanin in Substantia Nigra and Dystrophic Axons in Putamen of Parkinson's brain

Fibrillar amyloid β (fAβ) peptide is the major component of Aβ plaques in the brains of Alzheimer's disease (AD) patients. Inflammatory mediators have previously been proposed to be drivers of Aβ pathology in AD patients by increasing amyloidogenic processing of APP and promoting Aβ accumulation, but recent data have shown that expression of various inflammatory cytokines attenuates Aβ pathology in mouse models. In an effort to further study the role of different inflammatory cytokines on Aβ pathology in vivo, we explored the effect of murine Tumor Necrosis Factor α (mTNFα) in regulating Aβ accumulation. Recombinant adeno-associated virus serotype 1 (AAV2/1) mediated expression of mTNFα in the hippocampus of 4 month old APP transgenic TgCRND8 mice resulted in significant reduction in hippocampal Aβ burden. No changes in APP levels or APP processing were observed in either mTNFα expressing APP transgenic mice or in non-transgenic littermates. Analysis of Aβ plaque burden in mTNFα expressing mice showed that even after substantial reduction compared to EGFP expressing age-matched controls, the Aβ plaque burden levels of the former do not decrease to the levels of 4 month old unmanipulated mice. Taken together, our data suggests that proinflammatory cytokine expression induced robust glial activation can attenuate plaque deposition. Whether such an enhanced microglial response actually clears preexisting deposits without causing bystander neurotoxicity remains an open question.     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

The hypothalamus as the primary brain region of metabolic abnormalities in APP/PS1 transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) is an amyloid-related neurodegenerative disorder and is also considered to be a metabolic disease. Thus, investigation of metabolic mechanisms of amyloid pathology progression is of substantial importance for the diagnosis, prevention and treatment of AD. In the present study, cognitive function and brain metabolism were explored in the transgenic APP/PS1 mouse model of amyloid pathology at different ages. Using an NMR-based metabolomic approach, we examined metabolic changes in six different brain regions of wild-type and APP/PS1 mice at 1, 5 and 10 months of age. Learning and memory performance in mice was evaluated using the Morris water maze test. Furthermore, a generalized linear mixed model was employed to analyze the interaction effect between the mouse-type and brain region (or age) on metabolic alterations. Brain region-specific changes in energy metabolism occurred prior to a very early-stage of amyloid pathology (1 month of age) in APP/PS1 mice. A hypermetabolic state was identified in the brains of APP/PS1 mice at 5 months of age, and the hypothalamus was identified as the main brain region that underwent significant metabolic alterations. The cognitive function of APP/PS1 mice was impaired at 10 months of age; moreover, the hypermetabolic state identified in various brain regions at 5 months of age was also significantly decreased. In conclusion, our results suggest that a hypothalamic metabolism abnormality may comprise a potential indicator for the early-diagnosis and monitoring of amyloid pathology progression.     

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

Background

Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD.

Results

Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition.

Conclusions

The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis.