Transferrin-Conjugated Melittin-Loaded L-Arginine-Coated Iron Oxide Nanoparticles for Mitigating Beta-Amyloid Pathology of the 5XFAD Mouse Brain.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases and a major contributor to dementia. Although the cause of this condition has been identified long ago as aberrant aggregations of amyloid and tau proteins, effective therapies for it remain elusive. The complexities of drug development for AD treatment are often compounded by the impermeable blood-brain barrier and low-yield brain delivery. In addition, the use of high drug concentrations to overcome this challenge may entail side effects. To address these challenges and enhance the precision of delivery into brain regions affected by amyloid aggregation, we proposed a transferrin-conjugated nanoparticle-based drug delivery system. The transferrin-conjugated melittin-loaded L-arginine-coated iron oxide nanoparticles (Tf-MeLioNs) developed in this study successfully mitigated melittin-induced cytotoxicity and hemolysis in the cell culture system. In the 5XFAD mouse brain, Tf-MeLioNs remarkably reduced amyloid plaque accumulation, particularly in the hippocampus. This study suggested Tf-LioNs as a potential drug delivery platform and Tf-MeLioNs as a candidate for therapeutic drug targeting of amyloid plaques in AD. These findings provide a foundation for further exploration and advancement in AD therapeutics.

Effect Sizes of Cognitive and Locomotive Behavior Tests in the 5XFAD-J Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid ß (Aß) plaques in the brain, leading to cognitive impairment and other clinical symptoms. The 5XFAD mouse model is commonly used in AD research because it expresses five human transgenes that result in the accumulation of Aß plaques and cognitive decline at a relatively early age. Behavioral experiments are frequently conducted using this model; however, the effect size has not yet been reported. In this study, we examined basic cognition and locomotion in 5XFAD mice with a C57BL6/J background (5XFAD-J) at 6 months of age, a period in which impairments of cognitive function and locomotion are commonly observed. We analyzed the effect sizes of cognitive and locomotive experiments in the 5XFAD mice compared with those in the wild-type mice. Our results suggest that for long-term memory analysis, the novel object recognition test (p = 0.013, effect size 1.24) required a sample size of at least 12 to obtain meaningful results. Moreover, analysis of general locomotion over total distance with the Laboratory Animal Behavior Observation, Registration and Analysis System (LABORAS) test during the dark phase (p = 0.007, effect size -1.37) needed a sample size of 10 for a statistical power (1-ß) of 0.8. In conclusion, we can conduct more ethical and scientifically rigorous animal experiments using 5XFAD mice based on the effect and sample sizes suggested in this study.

Effect of Obesity and High-Density Lipoprotein Concentration on the Pathological Characteristics of Alzheimer's Disease in High-Fat Diet-Fed Mice.

The typical pathological features of Alzheimer's disease (AD) are the accumulation of amyloid plaques in the brain and reactivity of glial cells such as astrocytes and microglia. Clinically, the development of AD and obesity are known to be correlated. In this study, we analyzed the changes in AD pathological characteristics in 5XFAD mice after obesity induction through a high-fat diet (HFD). Surprisingly, high-density lipoprotein and apolipoprotein AI (APOA-I) serum levels were increased without low-density lipoprotein alteration in both HFD groups. The reactivity of astrocytes and microglia in the dentate gyrus of the hippocampus and fornix of the hypothalamus in 5XFAD mice was decreased in the transgenic (TG)-HFD high group. Finally, the accumulation of amyloid plaques in the dentate gyrus region of the hippocampus was also significantly decreased in the TG-HFD high group. These results suggest that increased high-density lipoprotein level, especially with increased APOA-I serum level, alleviates the pathological features of AD and could be a new potential therapeutic strategy for AD treatment.

Disruption of the astrocyte-neuron interaction is responsible for the impairments in learning and memory in 5XFAD mice: an Alzheimer's disease animal model.

The morphological dynamics of astrocytes are altered in the hippocampus during memory induction. Astrocyte-neuron interactions on synapses are called tripartite synapses. These control the synaptic function in the central nervous system. Astrocytes are activated in a reactive state by STAT3 phosphorylation in 5XFAD mice, an Alzheimer's disease (AD) animal model. However, changes in astrocyte-neuron interactions in reactive or resting-state astrocytes during memory induction remain to be defined. Here, we investigated the time-dependent changes in astrocyte morphology and the number of astrocyte-neuron interactions in the hippocampus over the course of long-term memory formation in 5XFAD mice. Hippocampal-dependent long-term memory was induced using a contextual fear conditioning test in 5XFAD mice. The number of astrocytic processes increased in both wild-type and 5XFAD mice during memory formation. To assess astrocyte-neuron interactions in the hippocampal dentate gyrus, we counted the colocalization of glial fibrillary acidic protein and postsynaptic density protein 95 via immunofluorescence. Both groups revealed an increase in astrocyte-neuron interactions after memory induction. At 24 h after memory formation, the number of tripartite synapses returned to baseline levels in both groups. However, the total number of astrocyte-neuron interactions was significantly decreased in 5XFAD mice. Administration of Stattic, a STAT3 phosphorylation inhibitor, rescued the number of astrocyte-neuron interactions in 5XFAD mice. In conclusion, we suggest that a decreased number of astrocyte-neuron interactions may underlie memory impairment in the early stages of AD.

Inhibition of STAT3 phosphorylation attenuates impairments in learning and memory in 5XFAD mice, an animal model of Alzheimer's disease.

The pathophysiological roles of astrocytes in the reactive state are thought to have important significance in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). However, the detailed mechanisms underlying the transition of astrocytes from the resting state to the reactive state during neurodegenerative disease largely remain to be defined. Here, we investigated the pathways involved in activating astrocytes from the resting state to the reactive state in primary cultured astrocytes treated with oligomeric Aß and in the hippocampus of 5XFAD mice. Treatment with oligomeric Aß induced an increase in reactive astrocytes, as assessed by the protein level of glial fibrillary acidic protein (GFAP) and this increase was caused by STAT3 phosphorylation in primary cultured astrocytes. The administration of Stattic, an inhibitor of STAT3, rescued the activation of astrocytes in primary cultured astrocytes and in the hippocampus of 6-month-old 5XFAD mice as well as impairments in learning and memory. Collectively, these results demonstrated that reactive astrocytes in the AD brain are induced via STAT3 and the impairments in learning and memory observed in 5XFAD mice are rescued by STAT3 inhibition, suggesting that the inhibition of STAT3 phosphorylation in astrocytes may be a novel therapeutic target for cognitive impairment in AD.

Fetal neural stem cells from a mouse model of 15q11-13 duplication syndrome exhibit altered differentiation into neurons and astrocytes.

The duplication of human chromosome 15q11-13 is known to be associated with an estimated 1.1% of autism cases. Here, we investigated whether differentiation into neurons and astrocytes is altered in fetal neural stem cells (FNSCs) isolated from the mouse model of 15q11-13 duplication syndrome (patDp/+ mice). In patDp/+ mice-derived FNSCs, multipotency was maintained for a longer period, the population of neurons was downregulated, and that of astrocytes was upregulated significantly after differentiation induction. These results suggest that the dysregulation of FNSCs differentiation could affect cortical development and behavioral deficits in the early postnatal stage shown in the patDp/+ mice.

Phloroglucinol ameliorates cognitive impairments by reducing the amyloid ß peptide burden and pro-inflammatory cytokines in the hippocampus of 5XFAD mice.

Among the various causative factors involved in the pathogenesis of Alzheimer's disease (AD), oxidative stress has emerged as an important factor. Phloroglucinol is a polyphenol component of phlorotannin, which is found at sufficient levels in Ecklonia cava (E. cava). Phloroglucinol has been reported to exert antioxidant activities in various tissues. Previously, we reported that the stereotaxic injection of phloroglucinol regulated synaptic plasticity in an AD mouse model. In this study, we aimed to investigate the effects of oral administration of phloroglucinol in AD. The oral administration of phloroglucinol for 2 months attenuated the impairments in cognitive function observed in 6-month-old 5X familial AD (5XFAD) mice, as assessed with the T-maze and Y-maze tests. The administration of phloroglucinol for 2 months in 5XFAD mice caused a reduction in the number of amyloid plaques and in the protein level of BACE1, a major amyloid precursor protein cleavage enzyme, together with γ-secretase. Phloroglucinol also restored the reduction in dendritic spine density and the number of mature spines in the hippocampi of 5XFAD mice. In addition, phloroglucinol-administered 5XFAD mice displayed lower protein levels of GFAP and Iba-1 and mRNA levels of TNF-α and IL-6 compared with vehicle-administered 5XFAD mice. These results demonstrated that phloroglucinol alleviated the neuropathological features and behavioral phenotypes in the 5XFAD mouse model. Taken together, our results suggest that phloroglucinol has therapeutic potential for AD treatment.

Dendritic spine anomalies and PTEN alterations in a mouse model of VPA-induced autism spectrum disorder.

Mounting evidence suggests that the etiology of autism spectrum disorders (ASDs) is profoundly influenced by exposure to environmental factors, although the precise molecular and cellular links remain ill-defined. In this study, we examined how exposure to valproic acid (VPA) during pregnancy is associated with an increased incidence of ASD. A mouse model was established by injecting VPA at embryonic day 13, and its behavioral phenotypes including impaired social interaction, increased repetitive behaviors and decreased nociception were observed at postnatal days 21-42. VPA-treated mice showed dysregulation of synaptic structure in cortical neurons, including a reduced proportion of filopodium-type and stubby spines and increased proportions of thin and mushroom-type spines, along with a decreased spine head size. We also found that VPA-treatment led to decreased expression of phosphate and tensin homolog (PTEN) and increased levels of p-AKT protein in the hippocampus and cortex. Our data suggest that there is a correlation between VPA exposure and dysregulation of PTEN with ASD-like behavioral and neuroanatomical changes, and this may be a potential mechanism of VPA-induced ASD.

Peg-Interferon Lambda Treatment Induces Robust Innate and Adaptive Immunity in Chronic Hepatitis B Patients.

IFN-lambda (IFNλ) is a member of the type III IFN family and is reported to possess anti-pathogen, anti-cancer, and immunomodulatory properties; however, there are limited data regarding its impact on host immune responses in vivo. We performed longitudinal and comprehensive immunosurveillance to assess the ability of pegylated (peg)-IFNλ to augment antiviral host immunity as part of a clinical trial assessing the efficacy of peg-IFNλ in chronic hepatitis B (CHB) patients. These patients were pretreated with directly acting antiviral therapy (entecavir) for 12 weeks with subsequent addition of peg-IFNλ for up to 32 weeks. In a subgroup of patients, the addition of peg-IFNλ provoked high serum levels of antiviral cytokine IL-18. We also observed the enhancement of natural killer cell polyfunctionality and the recovery of a pan-genotypic HBV-specific CD4+ T cells producing IFN-γ with maintenance of HBV-specific CD8+ T cell antiviral and cytotoxic activities. It was only in these patients that we observed strong virological control with reductions in both viral replication and HBV antigen levels. Here, we show for the first time that in vivo peg-IFNλ displays significant immunostimulatory properties with improvements in the main effectors mediating anti-HBV immunity. Interestingly, the maintenance in HBV-specific CD8+ T cells in the presence of peg-IFNλ is in contrast to previous studies showing that peg-IFNα treatment for CHB results in a detrimental effect on the functionality of this important antiviral T cell compartment. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01204762.

Dysfunction of Microglial STAT3 Alleviates Depressive Behavior via Neuron-Microglia Interactions.

Neuron-microglia interactions have a crucial role in maintaining the neuroimmune system. The balance of neuroimmune system has emerged as an important process in the pathophysiology of depression. However, how neuron-microglia interactions contribute to major depressive disorders has been poorly understood. Herein, we demonstrated that microglia-derived synaptic changes induced antidepressive-like behavior by using microglia-specific signal transducer and activator of transcription 3 (STAT3) knockout (KO) (STAT3fl/fl;LysM-Cre+/-) mice. We found that microglia-specific STAT3 KO mice showed antidepressive-like behavior in the forced swim, tail suspension, sucrose preference, and open-field tests. Surprisingly, the secretion of macrophage colony-stimulating factor (M-CSF) was increased from neuronal cells in the brains of STAT3fl/fl;LysM-Cre+/- mice. Moreover, the phosphorylation of antidepressant-targeting mediators and brain-derived neurotrophic factor expression were increased in the brains of STAT3fl/fl;LysM-Cre+/- mice as well as in neuronal cells in response to M-CSF stimulation. Importantly, the miniature excitatory postsynaptic current frequency in the medial prefrontal cortex was increased in STAT3fl/fl;LysM-Cre+/- mice and in the M-CSF treatment group. Collectively, microglial STAT3 regulates depression-related behaviors via neuronal M-CSF-mediated synaptic activity, suggesting that inhibition of microglial STAT3 might be a new therapeutic strategy for depression.

Alterations in protein phosphorylation in the amygdala of the 5XFamilial Alzheimer's disease animal model.

Alzheimer's disease is the most common disease underlying dementia in humans. Two major neuropathological hallmarks of AD are neuritic plaques primarily composed of amyloid beta peptide and neurofibrillary tangles primarily composed of hyperphosphorylated tau. In addition to impaired memory function, AD patients often display neuropsychiatric symptoms and abnormal emotional states such as confusion, delusion, manic/depressive episodes and altered fear status. Brains from AD patients show atrophy of the amygdala which is involved in fear expression and emotional processing as well as hippocampal atrophy. However, which molecular changes are responsible for the altered emotional states observed in AD remains to be elucidated. Here, we observed that the fear response as assessed by evaluating fear memory via a cued fear conditioning test was impaired in 5XFamilial AD (5XFAD) mice, an animal model of AD. Compared to wild-type mice, 5XFAD mice showed changes in the phosphorylation of twelve proteins in the amygdala. Thus, our study provides twelve potential protein targets in the amygdala that may be responsible for the impairment in fear memory in AD.

Hippocampus-based contextual memory alters the morphological characteristics of astrocytes in the dentate gyrus.

Astrocytes have been reported to exist in two states, the resting and the reactive states. Morphological changes in the reactive state of astrocytes include an increase in thickness and number of processes, and an increase in the size of the cell body. Molecular changes also occur, such as an increase in the expression of glial fibrillary acidic protein (GFAP). However, the morphological and molecular changes during the process of learning and memory have not been elucidated. In the current study, we subjected Fvb/n mice to contextual fear conditioning, and checked for morphological and molecular changes in astrocytes. 1 h after fear conditioning, type II and type III astrocytes exhibited a unique status with an increased number of processes and decreased GFAP expression which differed from the typical resting or reactive state. In addition, the protein level of excitatory excitatory amino acid transporter 2 (EAAT2) was increased 1 h to 24 h after contextual fear conditioning while EAAT1 did not show any alterations. Connexin 43 (Cx43) protein was found to be increased at 24 h after fear conditioning. These data suggest that hippocampus-based contextual memory process induces changes in the status of astrocytes towards a novel status different from typical resting or reactive states. These morphological and molecular changes may be in line with functional changes.

Phloroglucinol Attenuates the Cognitive Deficits of the 5XFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia among the elderly. Neuritic plaques whose primary component is amyloid beta peptide (Aß) and neurofibrillary tangles which are composed of hyperphosphorylated tau, are known to be the neuropathological hallmarks of AD. In addition, impaired synaptic plasticity in neuronal networks is thought to be important mechanism underlying for the cognitive deficits observed in AD. Although various causative factors, including excitotoxicity, mitochondrial dysregulation and oxidative damage caused by Aß, are involved in early onset of AD, fundamental therapeutics that can modify the progression of this disease are not currently available. In the present study, we investigated whether phloroglucinol (1, 3, 5-trihydroxybenzene), a component of phlorotannins, which are plentiful in Ecklonia cava, a marine brown alga species, displays therapeutic activities in AD. We found that phloroglucinol attenuates the increase in reactive oxygen species (ROS) accumulation induced by oligomeric Aß1-42 (Aß1-42) treatment in HT-22, hippocampal cell line. In addition, phloroglucinol was shown to ameliorate the reduction in dendritic spine density induced by Aß1-42 treatment in rat primary hippocampal neuron cultures. We also found that the administration of phloroglucinol to the hippocampal region attenuated the impairments in cognitive dysfunction observed in 22-week-old 5XFAD (Tg6799) mice, which are used as an AD animal model. These results indicate that phloroglucinol displays therapeutic potential for AD by reducing the cellular ROS levels.

Phloroglucinol attenuates motor functional deficits in an animal model of Parkinson's disease by enhancing Nrf2 activity.

In this study, we investigated whether phloroglucinol (1,3,5-trihydroxybenzene) has therapeutic effects in cellular and animal model of Parkinson's disease (PD). PD is the second most common, chronic and progressive neurodegenerative disease, and is clinically characterized with motor dysfunctions such as bradykinesia, rigidity, postural instability, gait impairment, and resting tremor. In the brains of PD patients, dopaminergic neuronal loss is observed in the Substantia nigra. Although the exact mechanisms underlying PD are largely unknown, mitochondrial dysfunction and oxidative stress are thought to be critical factors that induce the onset of the disease. Here, phloroglucinol administration was shown to attenuate motor functional deficits evaluated with rota-rod and apomorphine-induced rotation tests in 6-hydroxydopamine (6-OHDA)-induced PD animal models. Moreover, phloroglucinol ameliorated the loss of synapses as assessed with protein levels and immunoreactivity against synaptophysin in the midbrain region of the 6-OHDA-lesioned rats. In addition, in SH-SY5Y cultures, the cytotoxicity of 6-OHDA was reduced by pre-treatment with phloroglucinol. The increase in the reactive oxygen species, lipid peroxidation, protein carbonyl formation and 8-hydroxyguanine caused by treatment with 6-OHDA was attenuated by phloroglucinol in SH-SY5Y cells. Furthermore, phloroglucinol treatment rescued the reduced levels of nuclear Nrf2, antioxidant enzymes, i.e., catalase and glutathione peroxidase, in 6-OHDA-treated cells. Taken together, phloroglucinol has a therapeutic potential for treatment of PD.

Surface ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae).

The tegumental ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae) was observed by scanning electron microscopy. One- to 3-day-old juveniles and 10-day-old adults were harvested from chicks experimentally fed metacercariae from a bivalve, Mactra veneriformis. The juvenile worms were minute, curved ventrally, and had 23 collar spines characteristically arranged in a single row. The lips of the oral sucker had 7 single aciliated sensory papillae and 4 grouped uniciliated sensory papillae. The ventral sucker had 25 aciliated round swellings on its lip. The anterolateral surface between the 2 suckers was densely packed with tongue-shaped tegumental spines, and the ventral surface just posterior to the ventral sucker was covered with peg-like spines. Retractile, peg-like spines were seen on the anterolateral surface, whereas scale-like spines with round tips and broad bases were sparsely distributed posterior to the ventral sucker. The cirrus was characteristically protruding and armed with minute spines. The surface ultrastructure of A. tyosenense was unique, especially in the number and arrangement of collar spines, shape, and distribution of tegumental spines and in distribution of sensory papillae.