p38α‐MAPK‐deficient myeloid cells ameliorate symptoms and pathology of APP‐transgenic Alzheimer's disease mice

Alzheimer''s disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid‐β peptides (Aβ) and microglia‐dominated inflammatory activation in the brain. p38α‐MAPK is activated in both neurons and microglia. How p38α‐MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α‐MAPK in all myeloid cells or specifically in microglia of APP‐transgenic mice, and examined animals for AD‐associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α‐MAPK‐deficient myeloid cells were more effective than p38α‐MAPK‐deficient microglia in reducing cerebral Aβ and neuronal impairment in APP‐transgenic mice. Deficiency of p38α‐MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α‐MAPK‐deficient myeloid cells reduced IL‐17a‐expressing CD4‐positive lymphocytes in 9 but not 4‐month‐old APP‐transgenic mice. By cross‐breeding APP‐transgenic mice with Il‐17a‐knockout mice, we observed that IL‐17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9‐month‐old myeloid p38α‐MAPK‐deficient AD mice. Thus, p38α‐MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL‐17a‐expressing lymphocytes may partially mediate the pathogenic role of p38α‐MAPK in peripheral myeloid cells. Our study supports p38α‐MAPK as a therapeutic target for AD patients.     

Temporal and brain region‐specific elevations of soluble Amyloid‐β40‐42 in the Ts65Dn mouse model of Down syndrome and Alzheimer’s disease

Down syndrome (DS) is a leading cause of intellectual disability that also results in hallmark Alzheimer''s disease (AD) pathologies such as amyloid beta (Aβ) plaques and hyperphosphorylated tau. The Ts65Dn mouse model is commonly used to study DS, as trisomic Ts65Dn mice carry 2/3 of the triplicated gene homologues as occur in human DS. The Ts65Dn strain also allows investigation of mechanisms common to DS and AD pathology, with many of these triplicated genes implicated in AD; for example, trisomic Ts65Dn mice overproduce amyloid precursor protein (APP), which is then processed into soluble Aβ_40‐42 fragments. Notably, Ts65Dn mice show alterations to the basal forebrain, which parallels the loss of function in this region observed in DS and AD patients early on in disease progression. However, a complete picture of soluble Aβ_40‐42 accumulation in a region‐, age‐, and sex‐specific manner has not yet been characterized in the Ts65Dn model. Here, we show that trisomic mice accumulate soluble Aβ_40‐42 in the basal forebrain, frontal cortex, hippocampus, and cerebellum in an age‐specific manner, with elevation in the frontal cortex and hippocampus as early as 4 months of age. Furthermore, we detected sex differences in accumulation of Aβ_40‐42 within the basal forebrain, with females having significantly higher Aβ_40‐42 at 7–8 months of age. Lastly, we show that APP expression in the basal forebrain and hippocampus inversely correlates with Aβ_40‐42 levels. This spatial and temporal characterization of soluble Aβ_40‐42 in the Ts65Dn model allows for further exploration of the role soluble Aβ plays in the progression of other AD‐like pathologies in these key brain regions.     

γ‐Secretase modulators show selectivity for γ‐secretase–mediated amyloid precursor protein intramembrane processing

The aggregation of β‐amyloid peptide 42 results in the formation of toxic oligomers and plaques, which plays a pivotal role in Alzheimer''s disease pathogenesis. Aβ42 is one of several Aβ peptides, all of Aβ30 to Aβ43 that are produced as a result of γ‐secretase–mediated regulated intramembrane proteolysis of the amyloid precursor protein. γ‐Secretase modulators (GSMs) represent a promising class of Aβ42‐lowering anti‐amyloidogenic compounds for the treatment of AD. Gamma‐secretase modulators change the relative proportion of secreted Aβ peptides, while sparing the γ‐secretase–mediated processing event resulting in the release of the cytoplasmic APP intracellular domain. In this study, we have characterized how GSMs affect the γ‐secretase cleavage of three γ‐secretase substrates, E‐cadherin, ephrin type A receptor 4 (EphA4) and ephrin type B receptor 2 (EphB2), which all are implicated in important contexts of cell signalling. By using a reporter gene assay, we demonstrate that the γ‐secretase–dependent generation of EphA4 and EphB2 intracellular domains is unaffected by GSMs. We also show that γ‐secretase processing of EphA4 and EphB2 results in the release of several Aβ‐like peptides, but that only the production of Aβ‐like proteins from EphA4 is modulated by GSMs, but with an order of magnitude lower potency as compared to Aβ modulation. Collectively, these results suggest that GSMs are selective for γ‐secretase–mediated Aβ production.     

Physiological clearance of Aβ by spleen and splenectomy aggravates Alzheimer‐type pathogenesis

BackgroundA previous study demonstrated that nearly 40%–60% of brain Aβ flows out into the peripheral system for clearance. However, where and how circulating Aβ is cleared in the periphery remains unclear. The spleen acts as a blood filter and an immune organ. The aim of the present study was to investigate the role of the spleen in the clearance of Aβ in the periphery.MethodsWe investigated the physiological clearance of Aβ by the spleen and established a mouse model of AD and spleen excision by removing the spleens of APP/PS1 mice to investigate the effect of splenectomy on AD mice.ResultsWe found that Aβ levels in the splenic artery were higher than those in the splenic vein, suggesting that circulating Aβ is cleared when blood flows through the spleen. Next, we found that splenic monocytes/macrophages could take up Aβ directly in vivo and in vitro. Splenectomy aggravated behaviour deficits, brain Aβ burden and AD‐related pathologies in AD mice.ConclusionOur study reveals for the first time that the spleen exerts a physiological function of clearing circulating Aβ in the periphery. Our study also suggests that splenectomy, which is a routine treatment for splenic rupture and hypersplenism, might accelerate the development of AD.     

Activation of CREB‐mediated autophagy by thioperamide ameliorates β‐amyloid pathology and cognition in Alzheimer’s disease

Alzheimer''s disease (AD) is an age‐related neurodegenerative disease, and the imbalance between production and clearance of β‐amyloid (Aβ) is involved in its pathogenesis. Autophagy is an intracellular degradation pathway whereby leads to removal of aggregated proteins, up‐regulation of which may be a plausible therapeutic strategy for the treatment of AD. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Our previous study showed that thioperamide, as an antagonist of H3R, enhances autophagy and protects against ischemic injury. However, the effect of thioperamide on autophagic function and Aβ pathology in AD remains unknown. In this study, we found that thioperamide promoted cognitive function, ameliorated neuronal loss, and Aβ pathology in APP/PS1 transgenic (Tg) mice. Interestingly, thioperamide up‐regulated autophagic level and lysosomal function both in APP/PS1 Tg mice and in primary neurons under Aβ‐induced injury. The neuroprotection by thioperamide against AD was reversed by 3‐MA, inhibitor of autophagy, and siRNA of Atg7, key autophagic‐related gene. Furthermore, inhibition of activity of CREB, H3R downstream signaling, by H89 reversed the effect of thioperamide on promoted cell viability, activated autophagic flux, and increased autophagic‐lysosomal proteins expression, including Atg7, TFEB, and LAMP1, suggesting a CREB‐dependent autophagic activation by thioperamide in AD. Taken together, these results suggested that H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB‐mediated autophagy and lysosomal pathway, which contributed to Aβ clearance. This study uncovered a novel mechanism involving autophagic regulating behind the therapeutic effect of thioperamide in AD.     

Knockdown of astrocytic Grin2a aggravates β‐amyloid‐induced memory and cognitive deficits through regulating nerve growth factor

Synapse degeneration correlates strongly with cognitive impairments in Alzheimer''s disease (AD) patients. Soluble Amyloid‐beta (Aβ) oligomers are thought as the major trigger of synaptic malfunctions. Our earlier studies have demonstrated that Aβ oligomers interfere with synaptic function through N‐methyl‐D‐aspartate receptors (NMDARs). Our recent in vitro study found the neuroprotective role of astrocytic GluN2A in the promotion of synapse survival and identified nerve growth factor (NGF) derived from astrocytes, as a likely mediator of astrocytic GluN2A buffering against Aβ synaptotoxicity. Our present in vivo study focused on exploring the precise mechanism of astrocytic GluN2A influencing Aβ synaptotoxicity through regulating NGF. We generated an adeno‐associated virus (AAV) expressing an astrocytic promoter (GfaABC1D) shRNA targeted to Grin2a (the gene encoding GluN2A) to perform astrocyte‐specific Grin2a knockdown in the hippocampal dentate gyrus, after 3 weeks of virus vector expression, Aβ were bilaterally injected into the intracerebral ventricle. Our results showed that astrocyte‐specific knockdown of Grin2a and Aβ application both significantly impaired spatial memory and cognition, which associated with the reduced synaptic proteins PSD95, synaptophysin and compensatory increased NGF. The reduced astrocytic GluN2A can counteract Aβ‐induced compensatory protective increase of NGF through regulating pNF‐κB, Furin and VAMP3, which modulating the synthesis, mature and secretion of NGF respectively. Our present data reveal, for the first time, a novel mechanism of astrocytic GluN2A in exerting protective effects on synapses at the early stage of Aβ exposure, which may contribute to establish new targets for AD prevention and early therapy.     

Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

Inducing gamma oscillations with non‐invasive light flicker has been reported to impact Alzheimer''s disease‐related pathology. However, it is unclear which signaling pathways are involved in reducing amyloid load. Here, we found that gamma frequency light flicker increased anchoring of amyloid precursor protein (APP) to the plasma membrane for non‐amyloidogenic processing, and then physically interacted with KCC2, a neuron‐specific K⁺‐Cl⁻ cotransporter, suggesting that it is essential to maintain surface GABA_A receptor α1 levels and reduce β‐amyloid (Aβ) production. Stimulation with such light flicker limited KCC2 internalization and subsequent degradation via both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. Specifically, PKC‐dependent phosphorylation of APP on a serine residue was induced by gamma frequency light flicker, which was responsible for maintaining plasma membrane levels of full‐length APP, leading to its reduced trafficking to endosomes and inhibiting the β‐secretase cleavage pathway. The activated PKC from the gamma frequency light flicker subsequently phosphorylated serine of KCC2 and stabilized it onto the cell surface, which contributed to the upregulation of surface GABA_A receptor α1 levels. Together, these data indicate that enhancement of APP trafficking to the plasma membrane via light flicker plays a critical modulatory role in reduction of Aβ load in Alzheimer''s disease.     

3,6'‐disinapoyl sucrose attenuates Aβ1‐42‐ induced neurotoxicity in Caenorhabditis elegans by enhancing antioxidation and regulating autophagy

The aggregation of β‐amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer''s disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6''‐Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti‐depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ_1‐42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg‐laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ_1‐42 transgenic C. elegans. Gene analysis showed that DISS could up‐regulate the mRNA expression of sod‐3, gst‐4, daf‐16, bec‐1 and lgg‐1, while down‐regulate the mRNA expression of daf‐2 and daf‐15 in Aβ_1‐42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ_1‐42‐induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.     

GM‐CSF suppresses antioxidant signaling and drives IL‐1β secretion through NRF2 downregulation

GM‐CSF is a potent inflammatory cytokine regulating myeloid cell differentiation, hematopoiesis, and various other functions. It is functionally associated with a number of inflammatory pathologies including rheumatoid arthritis and inflammatory bowel disease. GM‐CSF has been found to promote NLRP3‐dependent IL‐1β secretion, which may have a significant role in driving inflammatory pathologies. However, the molecular mechanisms remain unknown. Here, we show that GM‐CSF induces IL‐1β secretion through a ROS‐dependent pathway. TNF is required for reactive oxygen species (ROS) generation that strikingly does not promote NLRP3 activation, but instead drives ubiquitylation of IL‐1β, promoting its cleavage through basal NRLP3 activity. GM‐CSF regulates this pathway through suppression of antioxidant responses via preventing upregulation of NRF2. Thus, the pro‐inflammatory effect of GM‐CSF on IL‐1β is through suppression of antioxidant responses, which leads to ubiquitylation of IL‐1β and enhanced processing. This study highlights the role of metabolic regulation of inflammatory signaling and reveals a novel mechanism for GM‐CSF to promote inflammation.     

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.     

Interplay between tau and α‐synuclein liquid–liquid phase separation

In Parkinson''s disease with dementia, up to 50% of patients develop a high number of tau‐containing neurofibrillary tangles. Tau‐based pathologies may thus act synergistically with the α‐synuclein pathology to confer a worse prognosis. A better understanding of the relationship between the two distinct pathologies is therefore required. Liquid–liquid phase separation (LLPS) of proteins has recently been shown to be important for protein aggregation involved in amyotrophic lateral sclerosis, whereas tau phase separation has been linked to Alzheimer''s disease. We therefore investigated the interaction of α‐synuclein with tau and its consequences on tau LLPS. We find α‐synuclein to have a low propensity for both, self‐coacervation and RNA‐mediated LLPS at pH 7.4. However, full‐length but not carboxy‐terminally truncated α‐synuclein efficiently partitions into tau/RNA droplets. We further demonstrate that Cdk2‐phosphorylation promotes the concentration of tau into RNA‐induced droplets, but at the same time decreases the amount of α‐synuclein inside the droplets. NMR spectroscopy reveals that the interaction of the carboxy‐terminal domain of α‐synuclein with the proline‐rich region P2 of tau is required for the recruitment of α‐synuclein into tau droplets. The combined data suggest that the concentration of α‐synuclein into tau‐associated condensates can contribute to synergistic aSyn/tau pathologies.     

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti‐tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL‐2 or IL‐15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA‐181a as a key modulator of human γδ T cell differentiation. We observe that miR‐181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR‐181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR‐181a overexpression restricts their levels of NKG2D and TNF‐α. Upon in silico analysis, we identify two miR‐181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR‐181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next‐generation immunotherapies.     

Direct interaction of β‐catenin with nuclear ESM1 supports stemness of metastatic prostate cancer

Wnt/β‐catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/β‐catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell‐specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/β‐catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of β‐catenin to stabilize β‐catenin–TCF4 complex and facilitate the transactivation of Wnt/β‐catenin signaling targets. Accordingly, activated β‐catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/β‐catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.     

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

Alzheimer''s disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

Phosphorylation of β‐catenin Ser60 by polo‐like kinase 1 drives the completion of cytokinesis

β‐Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that β‐catenin contributes to cytokinesis. Here, we identify a novel p‐S60 epitope on β‐catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of β‐catenin leads to cytokinesis‐defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of β‐catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between β‐catenin, Plk1, and Ect2. Time‐lapse image analysis confirmed the importance of β‐catenin phospho‐Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of β‐catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure‐derived human diseases.