Placenta-derived mesenchymal stem cells improve memory dysfunction in an Aβ1–42-infused mouse model of Alzheimer's disease

Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of central nervous system (CNS) and are currently being tested in clinical trials for neurological disorders, but preventive mechanisms of placenta-derived MSCs (PD-MSCs) for Alzheimer''s disease are poorly understood. Herein, we investigated the inhibitory effect of PD-MSCs on neuronal cell death and memory impairment in Aβ_1–42-infused mice. After intracerebroventrical (ICV) infusion of Aβ_1–42 for 14 days, the cognitive function was assessed by the Morris water maze test and passive avoidance test. Our results showed that the transplantation of PD-MSCs into Aβ_1–42-infused mice significantly improved cognitive impairment, and behavioral changes attenuated the expression of APP, BACE1, and Aβ, as well as the activity of β-secretase and γ-secretase. In addition, the activation of glia cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by the transplantation of PD-MSCs. Furthermore, we also found that PD-MSCs downregulated the release of inflammatory cytokines as well as prevented neuronal cell death and promoted neuronal cell differentiation from neuronal progenitor cells in Aβ_1–42-infused mice. These data indicate that PD-MSC mediates neuroprotection by regulating neuronal death, neurogenesis, glia cell activation in hippocampus, and altering cytokine expression, suggesting a close link between the therapeutic effects of MSCs and the damaged CNS in Alzheimer''s disease.     

Astrocytes are important mediators of A��-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer''s disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.     

Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain

Neurofibrillary tangles (NFT) and β-amyloid plaques are the neurological hallmarks of both Alzheimer''s disease and an unusual paralytic illness suffered by Chamorro villagers on the Pacific island of Guam. Many Chamorros with the disease suffer dementia, and in some villages one-quarter of the adults perished from the disease. Like Alzheimer''s, the causal factors of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) are poorly understood. In replicated experiments, we found that chronic dietary exposure to a cyanobacterial toxin present in the traditional Chamorro diet, β-N-methylamino-l-alanine (BMAA), triggers the formation of both NFT and β-amyloid deposits similar in structure and density to those found in brain tissues of Chamorros who died with ALS/PDC. Vervets (Chlorocebus sabaeus) fed for 140 days with BMAA-dosed fruit developed NFT and sparse β-amyloid deposits in the brain. Co-administration of the dietary amino acid l-serine with l-BMAA significantly reduced the density of NFT. These findings indicate that while chronic exposure to the environmental toxin BMAA can trigger neurodegeneration in vulnerable individuals, increasing the amount of l-serine in the diet can reduce the risk.     

Roles of receptor‐interacting protein kinase 1 in SH‐SY5Y cells with beta amyloid‐induced neurotoxicity

Alzheimer''s disease (AD), the major cause of dementia, affects the elderly population worldwide. Previous studies have shown that depletion of receptor‐interacting protein kinase 1 (RIPK1) expression reverted the AD phenotype in murine AD models. Necroptosis, executed by mixed lineage kinase domain‐like (MLKL) protein and activated by RIPK1 and RIPK3, has been shown to be involved in AD. However, the role of RIPK1 in beta‐amyloid (Aβ)‐induced necroptosis is not yet fully understood. In this study, we explored the role of RIPK1 in the SH‐SY5Y human neuroblastoma cells treated with Aβ 1–40 or Aβ 1–42. We showed that Aβ‐induced neuronal cell death was independent of apoptosis and autophagy pathways. Further analyses depicted that activation of RIPK1/MLKL‐dependant necroptosis pathway was observed in vitro. We demonstrated that inhibition of RIPK1 expression rescued the cells from Aβ‐induced neuronal cell death and ectopic expression of RIPK1 was found to enhance the stability of the endogenous APP. In summary, our findings demonstrated that Aβ can potentially drive necroptosis in an RIPK1‐MLKL‐dependent manner, proposing that RIPK1 plays an important role in the pathogenesis of AD.     

Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease

Alzheimer''s disease (AD) is a chronic neurodegenerative disease characterized by progressive neuronal loss and cognitive decline. Oligomeric amyloid β (oAβ) is involved in the pathogenesis of AD by affecting synaptic plasticity and inhibiting long-term potentiation. Although several lines of evidence suggests that microglia, the resident immune cells in the central nervous system (CNS), are neurotoxic in the development of AD, the mechanism whether or how oAβ induces microglial neurotoxicity remains unknown. Here, we show that oAβ promotes the processing of pro-interleukin (IL)-1β into mature IL-1β in microglia, which then enhances microglial neurotoxicity. The processing is induced by an increase in activity of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) via mitochondrial reactive oxygen species (ROS) and partially via NADPH oxidase-induced ROS. The caspase-1 inhibitor Z-YVAD-FMK inhibits the processing of IL-1β, and attenuates microglial neurotoxicity. Our results indicate that microglia can be activated by oAβ to induce neuroinflammation through processing of IL-1β, a pro-inflammatory cytokine, in AD.     

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (Aβ) in tissues with time. Aβ is a hallmark of Alzheimer''s disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy ^1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study Aβ-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human Aβ₄₂ into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing Aβ₄₂ is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.     

Clues to γ-secretase,huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum

Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT), the ataxins, the presenilins (PSEN1/PSEN2) are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene''s normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP) and presenilins, responsible for early onset Alzheimer''s disease (AD). To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will highlight recent studies on the function of HTT, presenilin γ-secretase and Hirano bodies conducted in Dictyostelium. I will then outline the limitations and future directions in using Dictyostelium to study disease, and finally conclude that given the evolutionary conservation of genes between Dictyostelium and humans and the organisms'' genetic tractability, that this system provides a fertile environment for discovering normal gene function related to neurodegeneration and will permit translational studies in higher systems.     

Neuronal loss and microgliosis are restricted to the core of Aβ deposits in mouse models of Alzheimer's disease

Amyloid‐β (Aβ) deposits, pathologic tau, and neurodegeneration are major pathological hallmarks of Alzheimer''s disease (AD). The relationship between neuronal loss and Aβ deposits is one of the fundamental questions in the pathogenesis of AD. However, this relationship is controversial. One main reason for the conflicting results may be the confounding effects of pathologic tau, which often coexists with Aβ deposits in the brains of AD patients. To clarify the relationship between neuronal loss and Aβ deposits, mouse models of AD, which develop abundant Aβ deposits in the aged brain without pathologic tau, were used to examine the co‐localization of NeuN‐positive neurons, NF‐H‐positive axons, MBP‐positive myelin sheaths, and Aβ deposits. Neuronal loss, as measured by decreased staining of the neuronal cell body, axon, and myelin sheath, as well as the IBA‐1‐positive microglia, was significantly increased in the core area of cerebral Aβ deposits, but not in adjacent areas. Furthermore, neuronal loss in the core area of cerebral Aβ deposits was correlated with Aβ deposit size. These results clearly indicate that neuronal loss is restricted to the core of Aβ deposits, and this restricted loss probably occurs because the Aβ deposit attracts microglia, which cluster in the core area where Aβ toxicity and neuroinflammation toxicity are restrained. These findings may contribute to our understanding of the relationship between neuronal loss and Aβ deposits in the absence of pathologic tau.     

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice

Blood–brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer''s disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer''s disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

Interaction between Aβ and Tau in the Pathogenesis of Alzheimer's Disease

Extracellular neuritic plaques composed of amyloid‑β (Aβ) protein and intracellular neurofibrillary tangles containing phosphorylated tau protein are the two hallmark proteins of Alzheimer''s disease (AD), and the separate neurotoxicity of these proteins in AD has been extensively studied. However, interventions that target Aβ or tau individually have not yielded substantial breakthroughs. The interest in the interactions between Aβ and tau in AD is increasing, but related drug investigations are in their infancy. This review discusses how Aβ accelerates tau phosphorylation and the possible mechanisms and pathways by which tau mediates Aβ toxicity. This review also describes the possible synergistic effects between Aβ and tau on microglial cells and astrocytes. Studies suggest that the coexistence of Aβ plaques and phosphorylated tau is related to the mechanism by which Aβ facilitates the propagation of tau aggregation in neuritic plaques. The interactions between Aβ and tau mediate cognitive dysfunction in patients with AD. In summary, this review summarizes recent data on the interplay between Aβ and tau to promote a better understanding of the roles of these proteins in the pathological process of AD and provide new insights into interventions against AD.     

COPS5 (Jab1) Protein Increases β Site Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation by Stabilizing RanBP9 Protein Levels

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer''s disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.     

Memory deficits in APP23/Abca1+/? mice correlate with the level of Aβ oligomers

ABCA1, a member of the ATP-binding cassette family of transporters, lipidates ApoE (apolipoprotein A) and is essential for the generation of HDL (high-density lipoprotein)-like particles in the CNS (central nervous system). Lack of Abca1 increases amyloid deposition in several AD (Alzheimer''s disease) mouse models. We hypothesized that deletion of only one copy of Abca1 in APP23 (where APP is amyloid precursor protein) AD model mice will aggravate memory deficits in these mice. Using the Morris Water Maze, we demonstrate that 2-year-old Abca1 heterozygous APP23 mice (referred to as APP23/het) have impaired learning during acquisition, and impaired memory retention during the probe trial when compared with age-matched wild-type mice (referred to as APP23/wt). As in our previous studies, the levels of ApoE in APP23/het mice were decreased, but the differences in the levels of Aβ and thioflavin-S-positive plaques between both groups were insignificant. Importantly, dot blot analysis demonstrated that APP23/het mice have a significantly higher level of soluble A11-positive Aβ (amyloid β protein) oligomers compared with APP23/wt which correlated negatively with cognitive performance. To confirm this finding, we performed immunohistochemistry with the A11 antibody, which revealed a significant increase of A11-positive oligomer structures in the CA1 region of hippocampi of APP23/het. This characteristic region-specific pattern of A11 staining was age-dependent and was missing in younger APP23 mice lacking Abca1. In contrast, the levels of Aβ*56, as well as other low-molecular-mass Aβ oligomers, were unchanged among the groups. Overall, the results of the present study demonstrate that in aged APP23 mice memory deficits depend on Abca1 and are likely to be mediated by the amount of Aβ oligomers deposited in the hippocampus.     

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.     

A Patient with Posterior Cortical Atrophy Possesses a Novel Mutation in the Presenilin 1 Gene

Posterior cortical atrophy is a dementia syndrome with symptoms of cortical visual dysfunction, associated with amyloid plaques and neurofibrillary tangles predominantly affecting visual association cortex. Most patients diagnosed with posterior cortical atrophy will finally develop a typical Alzheimer''s disease. However, there are a variety of neuropathological processes, which could lead towards a clinical presentation of posterior cortical atrophy. Mutations in the presenilin 1 gene, affecting the function of γ-secretase, are the most common genetic cause of familial, early-onset Alzheimer''s disease. Here we present a patient with a clinical diagnosis of posterior cortical atrophy who harbors a novel Presenilin 1 mutation (I211M). In silico analysis predicts that the mutation could influence the interaction between presenilin 1 and presenilin1 enhancer-2 protein, a protein partner within the γ-secretase complex. These findings along with published literature support the inclusion of posterior cortical atrophy on the Alzheimer''s disease spectrum.     

Cerebral microinfarct is emergency consequence of Alzheimer's disease: a new insight into development of neurodegenerative diseases

Imbalance of Aβ and tau protein production and clearance are the key factors among many causes of Alzheimer''s disease that leading to neurons degeneration and cognitive disorders. As a novel approach, glymphatic system quickly clear metabolic waste (especially Aβ and tau) from cerebral environment, and dysfunction of glymphatic system may relate to occurrence of Alzheimer''s disease. Microinfarct is a common histopathologic situation occurring in aging brain and leads to dramatic increase the generation of metabolic by-product after neuronal injury, hindering the operation of glymphatic system and suppress cerebral spinal fluid (CSF) and cerebral interstitial fluid (interstitial fluid, ISF) exchange. Microinfarcts destruct the integrity of microvascular and microstructural tissue, result in Aβ deposition and tau phosphorylation that form neurofibrillary tangles and associated with the cause of Alzheimer''s disease. Currently, it has been found that glymphatic system is involved in the pathological process of Alzheimer''s disease. Improving the function of glymphatic system after cerebral microinfarcts could be developed as a new approach for Alzheimer''s disease prevention and treatment. In this review, we will provide in-depth discussion on functional changes of glymphatic system after cerebral microinfarcts, further reveal pathogenesis of Alzheimer''s disease and provide a potentially more effective method for treatment of Alzheimer''s disease.     

Amyloid-β and Alzheimer's disease type pathology differentially affects the calcium signalling toolkit in astrocytes from different brain regions

The entorhinal–hippocampal circuit is severely affected in Alzheimer''s disease (AD). Here, we demonstrate that amyloid-β (Aβ) differentially affects primary cultured astrocytes derived from the entorhinal cortex (EC) and from the hippocampus from non-transgenic controls and 3xTg-AD transgenic mice. Exposure to 100 nM of Aβ resulted in increased expression of the metabotropic glutamate receptor type 5 (mGluR5) and its downstream InsP₃ receptor type 1 (InsP₃R1) in hippocampal but not in EC astrocytes. Amplitudes of Ca²⁺ responses to an mGluR5 agonist, DHPG, and to ATP, another metabotropic agonist coupled to InsP₃Rs, were significantly increased in Aβ-treated hippocampal but not in EC astrocytes. Previously we demonstrated that senile plaque formation in 3xTg-AD mice triggers astrogliosis in hippocampal but not in EC astrocytes. The different sensitivities of the Ca²⁺ signalling toolkit of EC versus hippocampal astrocytes to Aβ may account for the lack of astrogliosis in the EC, which in turn can explain the higher vulnerability of this region to AD.     

A familial Danish dementia rat shows impaired presynaptic and postsynaptic glutamatergic transmission

Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-β precursor protein (APP), whose mutations cause familial forms of Alzheimer''s disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2b^D rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid β (Aβ), a proteolytic product of APP, to AD, Itm2b^D rats were engineered to express two humanized App alleles and produce human Aβ. Here, we studied young Itm2b^D rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2b^D rats not only present subtle changes in human Aβ levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor–mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aβ and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia.