Involvement of the glymphatic/meningeal lymphatic system in Alzheimer's disease: insights into proteostasis and future directions.

BACKGROUND:  Alzheimer's disease (AD) is pathologically characterized by the abnormal accumulation of Aß and tau proteins. There has long been a keen interest among researchers in understanding how Aß and tau are ultimately cleared in the brain. The discovery of this glymphatic system introduced a novel perspective on protein clearance and it gained recognition as one of the major brain clearance pathways for clearing these pathogenic proteins in AD. This finding has sparked interest in exploring the potential contribution of the glymphatic/meningeal lymphatic system in AD. Furthermore, there is a growing emphasis and discussion regarding the possibility that activating the glymphatic/meningeal lymphatic system could serve as a novel therapeutic strategy against AD. OBJECTIVES:  Given this current research trend, the primary focus of this comprehensive review is to highlight the role of the glymphatic/meningeal lymphatic system in the pathogenesis of AD. The discussion will encompass future research directions and prospects for treatment in relation to the glymphatic/meningeal lymphatic system.

Chronic Neuronal Hyperexcitation Exacerbates Tau Propagation in a Mouse Model of Tauopathy.

The intracerebral spread of tau is a critical mechanism associated with functional decline in Alzheimer's disease (AD) and other tauopathies. Recently, a hypothesis has emerged suggesting that tau propagation is linked to functional neuronal connections, specifically driven by neuronal hyperactivity. However, experimental validation of this hypothesis remains limited. In this study, we investigated how tau propagation from the entorhinal cortex to the hippocampus, the neuronal circuit most susceptible to tau pathology in AD, is affected by the selective stimulation of neuronal activity along this circuit. Using a mouse model of seed-induced propagation combined with optogenetics, we found that the chronic stimulation of this neuronal connection over a 4-week period resulted in a significant increase in insoluble tau accumulation in both the entorhinal cortex and hippocampus. Importantly, the ratio of tau accumulation in the hippocampus relative to that in the entorhinal cortex, serving as an indicator of transcellular spreading, was significantly higher in mice subjected to chronic stimulation. These results support the notion that abnormal neuronal activity promotes tau propagation, thereby implicating it in the progression of tauopathy.

ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-ß pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-ß pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-ß pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

An adolescent and young adult (AYA) cancer education initiative aimed at healthcare students in Japan.

BACKGROUND: The focus on cancer in adolescents and young adults (AYA) has increased in recent years. We participated in an event called AYA week 2021 as part of a research project of the Ministry of Health, Labour and Welfare (MHLW) and conducted a fact-finding survey to collect information for raising public awareness of AYA-generation cancers and to improve support for AYA-generation cancer patients. METHODS: A 25-item questionnaire survey was conducted through requests sent to the deans of medical schools and presidents of related universities across Japan and by advertising on social networking sites and friendship networks. Furthermore, the effects of a lecture given by three cancer survivors on their cancer experience were examined. RESULTS: A total of 1288 healthcare students participated. The most common age group was between 20 and 24 years, with the majority being medical students (83%). The AYA cancer-educated population had more knowledge about AYA-generation cancers than the overall group. At present, very few people are familiar with AYA-generation cancers (30.5%), which highlights the importance of school education. There were 163 participants who attended the lecture given by the cancer survivors, of whom 108 completed the questionnaire. The results showed high participant satisfaction related to the lecture given by cancer survivors, suggesting that such lectures could help educate and raise awareness about AYA-generation cancers. CONCLUSIONS: The knowledge survey and lecture given by cancer survivors had educational effects. Many healthcare students responded positively, suggesting the efficacy of these types of initiatives.

Multifaceted Roles of Aquaporins in the Pathogenesis of Alzheimer's Disease.

The central nervous system is highly dependent on water, and disturbances in water homeostasis can have a significant impact on its normal functions. The regulation of water balance is, at least in part, carried out via specialized water channels called aquaporins. In the central nervous system, two major aquaporins (AQPs), AQP1 and AQP4, and their potential involvements have been long implicated in the pathophysiology of many brain disorders such as brain edema and Neuromyelitis optica. In addition to these diseases, there is growing attention to the involvement of AQPs in the removal of waste products in Alzheimer's disease (AD). This indicates that targeting fluid homeostasis is a novel and attractive approach for AD. This review article aims to summarize recent knowledge on the pathological implications of AQPs in AD, discussing unsolved questions and future prospects.

Amlexanox enhances the antitumor effect of anti-PD-1 antibody.

Cancer immunotherapy, especially treatment with monoclonal antibodies (mAbs) that block programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling, has attracted attention as a new therapeutic option for cancer. However, only a limited number of patients have responded to this treatment approach. In this study, we searched for compounds that enhance the efficacy of anti-PD-1 mAb using mixed lymphocyte reaction (MLR), which is a mixed culture system of the two key cells (dendritic and T cells) involved in tumor immunity. We found that amlexanox enhanced production of interferon (IFN)-γ, an indicator of T cell activation, by anti-PD-1 mAb. Amlexanox also induced PD-L1 expression in dendritic cells in MLR, whereas it did not stimulate interleukin-2 production by Jurkat T cells. These results suggest that amlexanox acts on dendritic cells, not T cells, in MLR. Furthermore, it enhanced the antitumor effect of the anti-PD-1 mAb in vivo in a mouse tumor-bearing model. The combination of amlexanox and anti-PD-1 mAb increased the expression of Ifng encoding IFN-γ, IFN-γ-related genes, Cd274 encoding PD-L1, and cytotoxic T cell-related genes in tumors. In conclusion, amlexanox stimulates the antitumor effect of anti-PD-1 mAb by acting on dendritic cells, which in turn activates cytotoxic T cells in tumors.

Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling.

Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with 13C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the 13C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.

[Causative Bacterial Strains and Sensitivityto Antimicrobial Agents in Female Acute Uncomplicated Cystitis].

Recently, wide spreading of fluoloquinolone resistant Escherichia coli is a serious problem inthe treatment of urinary tract infection. To investigate the causative bacterial strains of female acute uncomplicated cystitis (AUC) in the community and their sensitivity to antimicrobial agents, we retrospectively reviewed the medical records of 215 female AUC patients treated at our clinics from April 2014 to June 2015. Two hundred and nineteen strains were isolated as the causative bacteria from the patients'urine samples, including E. coli of 179 strains (82%) followed by Klebsiella pneumoniae (5.5%). One hundred and forty five strains (81%) of the isolated E. coli were sensitive to levofloxacin, whereas 32 strains (17.9%) were levofloxacin-resistant. To fosfomycin, the isolated E. coli showed the highest sensitivity (93.9%) among all antimicrobial agents tested. In univariate analysis, factors associated with levofloxacinresistant E. coli included two or more episodes of cystitis within the past year and levofloxacin use at the latest episode of cystitis. Inmultivariate analysis, two or more episodes of cystitis withinthe past year were found to be associated with levofloxacinresistan ce (p＝0.004). To prevent the increasing prevalence of infections caused by antibiotic-resistant bacteria, it is important to confirm the sensitivity of the causative agents for optimal antimicrobial therapy. The community-based surveillance data should be collected and considered when selecting empirical antimicrobial agents.

Antisense reduction of tau in adult mice protects against seizures.

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

A Method to Collect Cerebrospinal Fluid from Mouse Cisterna Magna to Determine Extracellular Tau Levels.

Despite being a cytoplasmic protein abundant in neurons, tau is detectable in various extracellular fluids. In addition to being passively released from dying/degenerating neurons, tau is also actively released from living neurons in a neuronal activity-dependent mechanism. In vivo, tau released from neurons first appears in brain interstitial fluid (ISF) and subsequently drains into cerebrospinal fluid (CSF) by glymphatic system. Changes in CSF tau levels alter during the course of AD pathogenesis and are considered to predict the disease-progression of AD. A method to collect CSF from various mouse models of AD will serve as a valuable tool to investigate the dynamics of physiological/pathological tau released from neurons. In this chapter, we describe and characterize a method that reliably collects a relatively large volume of CSF from anesthetized mice.

Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice.

Glymphatic system denotes a brain-wide pathway that eliminates extracellular solutes from brain. It is driven by the flow of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) via perivascular spaces. Glymphatic convective flow is driven by cerebral arterial pulsation, which is facilitated by a water channel, aquaporin-4 (AQP4) expressed in astrocytic end-foot processes. Since its discovery, the glymphatic system receives a considerable scientific attention due to its pivotal role in clearing metabolic waste as well as neurotoxic substances such as amyloid b peptide. Tau is a microtubule binding protein, however it is also physiologically released into extracellular fluids. The presence of tau in the blood stream indicates that it is eventually cleared from the brain to the periphery, however, the detailed mechanisms that eliminate extracellular tau from the central nervous system remained to be elucidated. Recently, we and others have reported that extracellular tau is eliminated from the brain to CSF by an AQP4 dependent mechanism, suggesting the involvement of the glymphatic system. In this chapter, we describe the detailed protocol of how we can assess glymphatic outflow of tau protein from brain to CSF in mice.

Late-onset Myoclonic Seizure in a 78-year-old Woman with Gaucher Disease.

We herein report a 78-year-old woman with Gaucher disease (GD) who was initially diagnosed with GD type 1, had been receiving long-term enzyme replacement therapy since 58 years old, and developed neurological manifestations in her 70s. The neurological manifestations included myoclonic seizures and progressive cognitive decline. Although it is rare for GD patients to first develop neurologic manifestations at such an advanced age, physicians engaged in long-term care for GD patients should be alert for this possibility.

Astrocytic APOE4 genotype-mediated negative impacts on synaptic architecture in human pluripotent stem cell model.

The APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer's disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined. In this study, we generated a human astrocyte model carrying the APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs) in which isogenic APOE4 iPSCs were genome edited from healthy control APOE3 iPSCs. Next, we demonstrated that the astrocytic APOE4 genotype negatively affects dendritic spine dynamics in a co-culture system with primary neurons. Transcriptome analysis revealed an increase of EDIL3, an extracellular matrix glycoprotein, in human APOE4 astrocytes, which could underlie dendritic spine reduction in neuronal cultures. Accordingly, postmortem AD brains carrying the APOE4 allele have elevated levels of EDIL3 protein deposits within amyloid plaques. Together, these results demonstrate the novel deleterious effect of human APOE4 astrocytes on synaptic architecture and may help to elucidate the mechanism of APOE4-linked AD pathogenesis.

In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice.

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Ternatin, a cyclic peptide isolated from mushroom, and its derivative suppress hyperglycemia and hepatic fatty acid synthesis in spontaneously diabetic KK-A(y) mice.

(-)-Ternatin is a highly methylated cyclic heptapeptide isolated from mushroom Coriolus versicolor. Ternatin has an inhibitory effect on fat accumulation in 3T3-L1 adipocytes. [D-Leu(7)]ternatin, a ternatin derivative, also inhibited fat accumulation in 3T3-L1 cells, although the effectiveness of [D-Leu(7)]ternatin was lower than that of ternatin. In this study, we investigated the effects of ternatin and [D-Leu(7)]ternatin on obesity and type 2 diabetes in KK-A(y) mice, an animal model for spontaneously developed type 2 diabetes. We continuously administered ternatin (8.5 or 17 nmol/day) or [D-Leu(7)]ternatin (68 nmol/day) to mice via a subcutaneous osmotic pump. Unexpectedly, neither ternatin nor [D-Leu(7)]ternatin affected body weight or adipose tissue weight in KK-A(y) mice. In contrast, it was demonstrated that both ternatin and [D-Leu(7)]ternatin suppress the development of hyperglycemia. In liver, the SREBP-1c mRNA level tended to be lower or significantly decreased in mice treated with ternatin or [D-Leu(7)]ternatin, respectively. Moreover, we found that ternatin directly lowered the SREBP-1c mRNA level in Hepa1-6 hepatocyte cells. This study showed that ternatin and [D-Leu(7)]ternatin each had a preventive effect on hyperglycemia and a suppressive effect on fatty acid synthesis in KK-A(y) mice.

NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary.

Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.

Isolation of 9-hydroxy-10E,12Z-octadecadienoic acid, an inhibitor of fat accumulation from Valeriana fauriei.

An EtOH extract of Valeriana fauriei was found to exhibit potent inhibition of fat accumulation against 3T3-L1 murine adipocytes. After performing several chromatographic steps, we successfully isolated the conjugated linoleic acid derivative, 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). Synthesized 9-HODE and its analogs showed inhibitory activity against fat accumulation.

Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration.

Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of ß-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE1). The ß-cleavage of APP by BACE1 is the initial and rate-limiting step in Aß generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The ß-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms.

APOE is the strongest genetic risk factor for late-onset Alzheimer's disease. ApoE exacerbates tau-associated neurodegeneration by driving microglial activation. However, how apoE regulates microglial activation and whether targeting apoE is therapeutically beneficial in tauopathy is unclear. Here, we show that overexpressing an apoE metabolic receptor, LDLR (low-density lipoprotein receptor), in P301S tauopathy mice markedly reduces brain apoE and ameliorates tau pathology and neurodegeneration. LDLR overexpression (OX) in microglia cell-autonomously downregulates microglial Apoe expression and is associated with suppressed microglial activation as in apoE-deficient microglia. ApoE deficiency and LDLR OX strongly drive microglial immunometabolism toward enhanced catabolism over anabolism, whereas LDLR-overexpressing microglia also uniquely upregulate specific ion channels and neurotransmitter receptors upon activation. ApoE-deficient and LDLR-overexpressing mice harbor enlarged pools of oligodendrocyte progenitor cells (OPCs) and show greater preservation of myelin integrity under neurodegenerative conditions. They also show less reactive astrocyte activation in the setting of tauopathy.